Electro-autotrophic synthesis of higher alcohols

ABSTRACT

The disclosure provides a process that converts CO 2  to higher alcohols (e.g. isobutanol) using electricity as the energy source. This process stores electricity (e.g. from solar energy, nuclear energy, and the like) in liquid fuels that can be used as high octane number gasoline substitutes. Instead of deriving reducing power from photosynthesis, this process derives reducing power from electrically generated mediators, either H 2  or formate. H 2  can be derived from electrolysis of water. Formate can be generated by electrochemical reduction of CO 2 . After delivering the reducing power in the cell, formate becomes CO 2  and recycles back. Therefore, the biological CO 2  fixation process can occur in the dark.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority under 35 U.S.C. §119 to U.S.Provisional Application No. 61/295,656, filed Jan. 15, 2010, thedisclosure of which is incorporated herein by reference.

BACKGROUND

Biofuels are an alternative for fossil fuels. For example, isobutanolcan be used as a high octane fuel for four-stroke internal combustionengines, as a pure component or in any portion as a mixture withgasoline. It has a high energy density (36 MJ/Kg) and low heat ofvaporization (0.43 MJ/Kg), both of which satisfy the requirements(energy density ≧32 MJ/Kg, heat of vaporization <0.5 MJ/Kg) specified bythis FOA. The research octane number of isobutanol is 110, which alsosatisfies the requirement (>85).

SUMMARY

The disclosure provides recombinant microorganisms that take advantageof the biological C—C bond formation capability without relying oninefficient photoenergy conversion (see, e.g., FIG. 1). Instead,reducing power is generated from electricity (including sunlight) todrive the metabolic process that forms C—C bonds necessary for liquidfuel synthesis. Thus, the microorganism of the disclosure utilizesman-made photoconversion and the biological C—C bond synthesis to makeliquid fuels. The pathways engineered into microorganisms as describedherein utilize electrically generated reducing mediators (H₂ or formate)to drive the “dark reaction” of CO₂ fixation. Both H₂ and formate can beused to reduce NAD(P)+ to NAD(P)H, which is then used as the reducingequivalent in CO₂ reduction, fuel synthesis, and ATP synthesis (FIG.1C). Once CO₂ is fixed in a metabolic intermediate, such as pyruvate, itcan be diverted to make isobutanol and other biofuels. The biologicalprocesses (H₂ or formate utilization, CO₂ fixation, fuel synthesis) canbe independently or all engineered into the same cell so long as thepathway comprises CO₂ fixation and utilizes reducing mediators alongwith the specific biofuel pathway. Furthermore, bioreactors andelectrolysis units can be integrated to form an electro-bio reactionunit.

The disclosure provides a recombinant microorganism capable of using H₂or formate for reduction of CO₂ and wherein the microorganism producesan alcohol selected from the group consisting of 1-propanol, isobutanol,1-butanol, 2-methyl 1-butanol, 3-methyl 1-butanol and 2-phenylethanolfrom CO₂ as the carbon source, wherein the alcohol is produced from ametabolite comprising a 2-keto acid. In one embodiment, themicroorganism has a naturally occurring H₂ and/or formate reductionpathway and at least one recombinant enzyme for the production of anintermediate in the synthesis of the alcohol. In another embodiment, themicroorganism comprises expression of a heterologous or overexpressionof an endogenous carbon-fixation enzyme and heterologous oroverexpression of a hydrogenase and/or formate dehydrogenase such thatthe microorganism can utilize H₂ and/or formate as a reducingmetabolite. In any of the foregoing embodiments, the alcohol can beisobutanol. In yet another embodiment, the recombinant microorganism isobtained from a Ralstonia sp. parental organism. In another embodiment,the 2-keto acid is selected from the group consisting of 2-ketobutyrate,2-ketoisovalerate, 2-ketovalerate, 2-keto 3-methylvalerate, 2-keto4-methyl-pentanoate, and phenylpyruvate. In one embodiment, themicroorganism comprises elevated expression or activity of a 2-keto-aciddecarboxylase and an alcohol dehydrogenase, as compared to a parentalmicroorganism. In one embodiment, the 2-keto-acid decarboxylase isselected from the group consisting of Pdc6, Aro10, Thi3, Kivd, and Pdc,or homolog thereof. In yet another embodiment, the 2-keto-aciddecarboxylase is encoded by a nucleic acid sequence derived from a geneselected from the group consisting of PDC6, ARO10, THI3, kivd, and pdc,or homolog thereof. In a specific embodiment, the 2-keto-aciddecarboxylase is encoded by a nucleic acid sequence derived from thekivd gene, or homolog thereof. In one embodiment, the alcoholdehydrogenase is Adh2, or homolog thereof. In another embodiment, thealcohol dehydrogenase is encoded by a nucleic acid sequence derived fromthe ADH2 gene, or homolog thereof. In another embodiment, themicroorganism is selected from a genus of Escherichia, Corynebacterium,Lactobacillus, Lactococcus, Salmonella, Enterobacter, Enterococcus,Erwinia, Pantoea, Morganella, Pectobacterium, Proteus, Ralstonia,Serratia, Shigella, Klebsiella, Citrobacter, Saccharomyces, Dekkera,Klyveromyces, and Pichia. In one embodiment, not only does the organismcomprise a pathway for utilizing H2 or formate but the organism also hasa modification in the biosynthetic pathway for the production of anamino acid to produce the alcohol. The microorganism can also havereduced ethanol production capability compared to a parentalmicroorganism. For examples, the microorganism comprises a reduction orinhibition in the conversion of acetyl-coA to ethanol. The microorganismcan comprise a reduction of an ethanol dehydrogenase thereby providing areduced ethanol production capability. In specific embodiments of any ofthe foregoing the microorganism produces greater than 100 mg/L ofisobutanol in 40 hours from sugar. In another specific embodiments ofany of the foregoing, the microorganism produces greater than 150 mg/Lof 3-methyl-1-butanol in 40 hours from sugar. In another embodiment, themicroorganism produces 120 mg/L of isobutanol or 180 mg/L of3-methyl-1-butanol.

The disclosure also provides a method of producing a biofuel, comprisingculturing a microorganism of any of the foregoing embodiments underconditions and in the presence or a suitable carbon source and reducingagent and isolating the biofuel. In one embodiment, the biofuel isisobutanol. In another embodiment, the reducing agent is formate or H₂.In yet a further embodiment, the microorganism is obtained from aRalstonia sp. parental organism.

The disclosure also provides a bioreactor system comprising a source ofH2 or formate, a source of energy to generate H2 or a combinationthereof, a source of CO2 and a recombinant microorganism of thedisclosure. In one embodiment, the disclosure can comprise a lightsource for photosynthesis.

DESCRIPTION OF THE FIGURES

FIG. 1A-F shows various pathways described in the disclosure. A) showsCO₂ fixation to produce pyruvate via the CBB cycle. B) shows a generalpathway for producing isobutanol from pyruvate. C) shows anelectro-autotrophic pathway of the disclosure. D) shows various pathwaysfor the production of biofuels. E) shows production of various ketoacids from pyruvate. F) shows valine biosynthetic pathways used inRalstonia eutropha.

FIG. 2A-G shows isobutyraldehyde, isobutanol production and cell growthin microorganism of the disclosure. FIGS. 2A-B show production directlyfrom CO₂ using engineered cyanobacterium S. elogatus. A. cumulativeproduction of isobutyraldehyde and B. Daily production ofisobutyraldehyde. C. shows characterization of the enhanced enzymeactivities for 2-KIV production. D. shows expression system for 2-KIVconversion into isobutanol. E. shows autotrophic production ofisobutanol using recombinant Ralstonia eutropha LH74. F. shows theeffect of different AHAS genes on isobutanol production in Ralstonia. G.shows autotrophic growth fo R. eutropha H16 on formate.

FIG. 3 depicts a nucleic acid sequence (SEQ ID NO:1) derived from a kivdgene encoding a polypeptide having 2-keto-acid decarboxylase activity.

FIG. 4 depicts a nucleic acid sequence (SEQ ID NO:3) derived from a PDC6gene encoding a polypeptide having 2-keto-acid decarboxylase activity.

FIG. 5 depicts a nucleic acid sequence (SEQ ID NO:5) derived from anARO10 gene encoding a polypeptide having 2-keto-acid decarboxylaseactivity.

FIG. 6 depicts a nucleic acid sequence (SEQ ID NO:7) derived from a THI3gene encoding a polypeptide having 2-keto-acid decarboxylase activity.

FIG. 7 depicts a nucleic acid sequence (SEQ ID NO:9) derived from a pdcgene encoding a polypeptide having 2-keto-acid decarboxylase activity.

FIG. 8 depicts a nucleic acid sequence (SEQ ID NO:11) derived from anADH2 gene encoding a polypeptide having alcohol dehydrogenase activity.

FIG. 9 depicts a nucleic acid sequence (SEQ ID NO:13) derived from ani/v/gene encoding a polypeptide having acetolactate synthase largesubunit activity.

FIG. 10 depicts a nucleic acid sequence (SEQ ID NO:15) derived from anilvH gene encoding a polypeptide having acetolactate synthase smallsubunit activity.

FIG. 11 depicts a nucleic acid sequence (SEQ ID NO:17) derived from anilvC gene encoding a polypeptide having acetohydroxy acidisomeroreductase activity.

FIG. 12 depicts a nucleic acid sequence (SEQ ID NO:19) derived from anilvD gene encoding a polypeptide having dihydroxy-acid dehydrataseactivity.

FIG. 13 depicts a nucleic acid sequence (SEQ ID NO:21) derived from anilvA gene encoding a polypeptide having threonine dehydratase activity.

FIG. 14 depicts a nucleic acid sequence (SEQ ID NO:23) derived from aleuA gene encoding a polypeptide having 2-isopropylmalate synthaseactivity.

FIG. 15 depicts a nucleic acid sequence (SEQ ID NO:25) derived from aleuB gene encoding a polypeptide having beta-isopropylmalatedehydrogenase activity.

FIG. 16 depicts a nucleic acid sequence (SEQ ID NO:27) derived from aleuC gene encoding a polypeptide having isopropylmalate isomerase largesubunit activity.

FIG. 17 depicts a nucleic acid sequence (SEQ ID NO:29) derived from aleuD gene encoding a polypeptide having isopropylmalate isomerase smallsubunit activity.

FIG. 18 depicts a nucleic acid sequence (SEQ ID NO:31) derived from acimA gene encoding a polypeptide having alpha-isopropylmalate synthaseactivity.

FIG. 19 depicts a nucleic acid sequence (SEQ ID NO:33) derived from anilvM gene encoding a polypeptide having acetolactate synthase largesubunit activity.

FIG. 20 depicts a nucleic acid sequence (SEQ ID NO:35) derived from anilvG gene encoding a polypeptide having acetolactate synthase smallsubunit activity.

FIG. 21 depicts a nucleic acid sequence (SEQ ID NO:37) derived from anilvN gene encoding a polypeptide having acetolactate synthase largesubunit activity.

FIG. 22 depicts a nucleic acid sequence (SEQ ID NO:39) derived from anilvB gene encoding a polypeptide having acetolactate synthase smallsubunit activity.

FIG. 23 depicts. a nucleic acid sequence (SEQ ID NO:41) derived from anadhE2 gene encoding a polypeptide having alcohol dehydrogenase activity.

FIG. 24 depicts a nucleic acid sequence (SEQ ID NO:43) derived from aLi-cimA gene encoding a polypeptide having alpha-isopropylmalatesynthase activity.

FIG. 25 depicts a nucleic acid sequence (SEQ ID NO:45) derived from aLi-leuC gene encoding a polypeptide having isopropylmalate isomeraselarge subunit activity.

FIG. 26 depicts a nucleic acid sequence (SEQ ID NO:47) derived from aLi-leuD gene encoding a polypeptide having isopropylmalate isomerasesmall subunit activity.

FIG. 27 depicts a nucleic acid sequence (SEQ ID NO:49) derived from aLi-leuB gene encoding a polypeptide having beta-isopropylmalatedehydrogenase activity.

FIG. 28 depicts a nucleic acid sequence (SEQ ID NO:51) derived from apheA gene encoding a polypeptide having chorismate mutase P/prephenatedehydratase activity.

FIG. 29 depicts a nucleic acid sequence (SEQ ID NO:53) derived from aTyrA gene encoding a polypeptide having chorismate mutase T/prephenatedehydratase activity.

FIG. 30 depicts a nucleic acid sequence (SEQ ID NO:55) derived from analsS gene encoding a polypeptide having acetolactate synthase activity.

DETAILED DESCRIPTION

As used herein and in the appended claims, the singular forms “a,”“and,” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a microorganism”includes a plurality of such microorganisms and reference to “thepolypeptide” includes reference to one or more polypeptides known tothose skilled in the art, and so forth.

Also, the use of “or” means “and/or” unless stated otherwise. Similarly,“comprise,” “comprises,” “comprising” “include,” “includes,” and“including” are interchangeable and not intended to be limiting.

It is to be further understood that where descriptions of variousembodiments use the term “comprising,” those skilled in the art wouldunderstand that in some specific instances, an embodiment can bealternatively described using language “consisting essentially of” or“consisting of”

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this disclosure belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice of the disclosed methods and compositions, the exemplarymethods, devices and materials are described herein.

The publications discussed above and throughout the text are providedsolely for their disclosure prior to the filing date of the presentapplication. Nothing herein is to be construed as an admission that theinventors are not entitled to antedate such disclosure by virtue ofprior disclosure.

The fixation of CO₂ into longer chain chemicals suitable for use asliquid fuels requires 1) formation of C—C bond, and 2) reduction ofcarbon. In plants and photosynthetic microorganisms, CO₂ fixation (thedark reaction) is coupled with the light reaction of photosynthesis,which produces the reducing power (NADPH) and energy (ATP). However, invarious photosynthetic systems light penetration in culture environmentscan be limiting, reducing efficiency and fuel production.

Nature has evolved organisms that have decoupled the photosynthesisprocess required for producing reducing power. A group of microbesderive energy and reducing power from chemicals (chemoautotrophs) suchas formate, or inorganics (lithoautotrophs) such as H₂ to drive CO₂fixation. Examples of these organisms include Ralstonia (formerlyAlcaligenes) and Xanthobacter. In particular, Ralstonia eutropha hasbeen extensively studied for the production of polyhydroxyalkanoate(PHA) industrially. It is metabolically active and versatile, and growsreasonably fast. Ralstonia can use either H₂ or formate to drive CO₂fixation through the CBB cycle. These organisms have hydrogenases andformate dehydrogenase to derive NAD(P)H from H₂ and formate,respectively. Thus, the NAD(P)H and ATP that are needed to drive CO₂fixation are obtained either via the CBB or rTCA cycles. For example,NADH can be derived from H₂ via hydrogenases or formate via formatedehydrogenases. NADH can then be converted to NADPH viatranshydrogenases. ATP is generated via the electron transport chainusing O₂ as the terminal electron acceptor.

The disclosure provides methods and compositions for the production ofhigher alcohols using a culture of microorganisms that utilizes CO₂ as acarbon source and utilizes a non-light or light and non-light producedreducing agent for production of NADPH (e.g., chemoautotrophs,lithoautotrophes, photoautotrophs and any combination thereof).

The disclosure utilizes recombinant micoorganisms and engineeredmetabolic pathways for microbial production of higher alcohols. Thesepathways can be engineered into E. coli, Saccharomyces cerevisiae,Bacillus subtilis, Clostridia, Ralstonia (formerly Alcaligenes),Xanthobacter and Corynebacteria.

Examples of microorganisms that utilize CO₂ as a carbon source includephotoautotrophs, chemoautotrophs and lithoautotrophs. In someembodiments, that methods and compositions comprise a co-culture ofautotrophs, photoautotrophs and a photoheterotroph or a photoautotrophand a microorganism that cannot utilize CO₂ as a carbon source.

S. elongatus does not utilize H₂ or formate as an electron donor. In oneembodiment, the disclosure provides recombinant microorganisms thatcomprise an engineered pathway (e.g., comprising a hydrogenase orformate dehydrogenase) to utilize H₂ or formate as an electron donor. Inone embodiment, S. elongatus is engineered to utilize these electronsources and alter its innate regulation networks to fix CO₂ in the dark.On the other hand, Ra. eutropha and Rh. palustris are able to utilize H₂or formate as electron sources to fix CO₂ in the dark. In theseorganisms, a biofuel production pathway that converts pyruvate or othersuitable intermediate into the biofuel (e.g., isobutanol) is engineeredinto these microorganisms in an efficient way.

E. coli, for example, has three hydrogenases, of which at least onehydrogenase has been shown to be reversible. By using the nativereversible hydrogenase of E. coli under high pressure of hydrogen in theculture or by overexpressing hydrogenases from other species (e.g., Ra.eutropha), E. coli can be engineered to harness the power of hydrogenaseto use hydrogen as an energy source.

The disclosure can utilize such parental organisms with heterologouspolynucleotides to promote the biosynthetic pathway for the productionof biofuels. In one embodiment, Ralstonia eutropha is used as a hostorganism for isobutanol production. In other embodiments, the disclosureprovides a recombinant microorganism that comprises a heterologous CO₂fixation enzyme and a non-light producing reducing agent.

H₂ and formate are used as exemplary reducing mediators. H₂ can begenerated from water hydrolysis, and formate can be generated byelectrochemical reduction of CO₂. The former process has beenextensively studied and industrial processes have been developed.Formate can be used as the electron mediator to circumvent the safetyissue of H₂ utilization. H₂ can be transferred to the microbes, and thereducing power can be extracted by hydrogenase to drive the CO₂ fixationprocess. Formate can also be taken up by cells and produce NAD(P)H andCO₂ by formate dehydrogenase. NAD(P)H is then used to drive CO₂fixation. O₂ is chosen as the terminal electron acceptor, as it is mostenvironmentally friendly. Any other electron acceptors will cause toomuch environmental upset to be scalable.

The yield of isobutanol from sugar has already reached industrial level.Since the pathways developed take advantage of the keto acid chemistry,which is used in amino acid biosynthesis, they are readily compatiblewith many organisms. Furthermore, the pathway platform has beenengineered in a photosynthetic microorganism, Synechococcus elongatusPCC7942, to produce isobutyraldehyde and isobutanol directly from CO₂(see, e.g., FIG. 1). The engineered strain produced isobutanol with aproduction rate higher than those reported for ethanol, hydrogen, orlipid production from cyanobacteria or algae (FIG. 2).

This disclosure demonstrates that alternative reducing processes, otherthan photosynthesis light reactions, can be used. For example, H₂,formate and electricity can be used instead of photosynthesis to deliverchemical reducing power to drive CO₂ fixation using theCalvin-Benson-Bassham (CBB) cycle and the biosynthesis of isobutanol.The chemical redox mediator (H₂ or formate). H₂ and formate can beevenly distributed in a large volume to promote redox avoiding problemsassociated with light-penetration associated with photosynthesis.

H₂ and formic acid can be used as the reducing mediators. The selectionwill depend on efficiency of the overall process. H₂ can be generatedfrom water hydrolysis, and formic acid can be generated byelectrochemical reduction of CO₂. Both of these processes have beenextensively studied and industrial processes have been developed. Theefficiencies of these processes are similar. H₂ can be transferred tothe microbes, and the reducing power can be extracted by hydrogenase todrive the CO₂ fixing process. Formic acid is the primary product of CO₂reduction electrochemically with the highest current efficiency. It canalso be taken up by cells and produce NAD(P)H and CO, by formatedehydrogenase. NAD(P)H is then used to drive CO₂ fixation.

The generation of H₂ and biochemical utilization are relativelystraightforward. These two redox mediators are relatively inexpensiveand can be dispersed in large volumes without high surface areas. H₂ andformate are produced from water and CO₂, respectively, and they arecycled back as such.

Competing alternatives include i) direct electrode coupling to cellssuch as Geobactor, ii) metal ions as mediators, iii) other organiccompounds as mediators. Direct electrode coupling requires highelectrode surface areas to drive the slow biological reaction.

The cyanobacterium, S. elongates, can be engineered to accept H₂ andformate as electron donors, and to decouple the CBB cycle from the lightreaction. The advantage of cyanobacteia is that they can also harvestsun light and thus can use photosynthesis wherever light is availableand use reducing mediator wherever light is unavailable. This strategyallows the organism to use both solar energy directly or indirectlythrough mediators and solves the problem of large light area requirementof photosynthesis. Another advantage of cyanobacteria is that synthesisof isobutanol and isobutyraldehyde can be achieved in relatively highproductivity.

For example, CO₂ is converted to pyruvate, which is then converted toisobutanol via the keto acid pathway (FIG. 4). AlsS (from B. subtilis)and ilvCD (from E. coli), and kivd (from Lactococcus lactis) are themost effective in producing isobutanol and isobutyraldehyde, from ketoacids and can be readily expressed in multiple organisms. These genescan be used initially to achieve isobutanol production.

The overall reaction of CO₂ fixation to isobutanol via the CBB cycle iscalculated as follows:

6CO₂+12NADPH+14ATP→Isobutanol+12NADP+14ADP+2CO₂

The ATP expenditure is slightly better than the CO₂ production toglucose on a per carbon basis.

The CBB cycle is the most common and best studied pathway for CO₂fixation. However, its energy expenditure is the highest, because ituses the high energy phospho-group to activate intermediates. Othercompeting pathways include the Wood-Ljundahl (reductive acetyl coA)pathway, the reductive TCA cycle, the 3-hydroxypropionate(3HP)/glyoxylate cycle, and the 3HP/4-hydroxybutyrate (4HP) cycle.

The overall reducing equivalent requirement and ATP equivalentrequirement of each pathway are summarized in Table 1. Note that thesepathways all have the same requirement for reducing equivalent, as it isdictated by the chemical structures of the substrate and the product.However, CBB and 3HP/glyoxylate are the most energy intensive, while thereductive TCA and Wood-Ljundahl pathways are most energy efficient. Ifthe P/0 ratio is assumed to be 2, the total reducing equivalent requiredby using CBB, pathway is 19, while the reduced TCA or Wood-Ljundahlpathways use 14 and 13 total reducing equivalents, respectively. Theenergy saving by using these more efficient pathways amounts to 26-30%.

TABLE 1 Reducing equivalent “[H₂]” and ATP equivalent “~P” needed foreach CO₂ fixing pathway. “[H₂]” represents a two-electron donor, such asNAD(P)H, Flavin-H₂, or 2 reduced Ferredoxins. Total “[H₂]” = “[H₂]” +“~P”/2, with an assumption that P/O ration equals 2. Pathways CO₂ H₂CO₃“[H₂]” “~P” Total “[H₂]” CBB 6 0 12 14 19 3HP/glyoxylate 0 6 12 14 193HP/4HB 2 4 12 12 18 reductive TCA 6 0 12 4 14 Wood-Ljundahl 6 0 12 2 13

However, other pathways are typically used by thermophiles (Table 2).

TABLE 2 Comparison of different CO₂ fixation organisms litho/chemoexisting growth O2 doubling genetic Pathways Organisms autotrophic?electon donor temp sensitive? time tools Comments CBB Synechococcus tobe photosynthesis 30 C. no    4 h available produce isobtuanol elongatusengineered Ralstonia yes H2, Formate 30 C. no  5-10 h available producePHA eutropha Reductive TCA Hydrogenobacter yea H2 70 C. no   15 h no lowdensity culture thermophilus Chlorobium yes thiosulfate 26-29 C.   yes15-20 h no low density culture limicola Wood-Ljundahl Moorella yes H2,formate 55-60 C.   somewhat 15-20 h no low density culture thermoacetica

For the above reasons, suitable hosts includes, for example,cyanobacteria, S. elongates and R. eutropha. R. eutropha can already useH₂ and formate as electron donors for CO₂ fixation, and has been usedindustrially for PHA synthesis. Its growth rate is acceptable andgenetic tools are available. The isobutanol pathway genes (FIG. 4) canbe expressed in R. eutropha to produce isobutanol from CO₂ and H₂ andformate. S. elongates has been used for isobutanol production from CO₂with high productivity. S. elongates can be engineered to use H₂ orformate as electron donors by expressing hydrogenase and formatedehydrogenase. The organism can also be engineered to further inactivateinnate regulations that coordinate the light reaction with the darkreaction. The resulting organism can use either light or electronmediators (H₂ or formate) to drive isobutanol production from CO₂.

In the recombinant microorganisms of the disclosure the CBB pathwaygenes are amplified and deregulated so that they are not subject totranscription level or protein level control. The use of electronmediators in low O₂ environment also reduces photorespiration ofRubisco, which is a major efficiency loss in photosynthesis.Ribulose-1,5-bisphosphate carboxylase oxygenase, most commonly known bythe shorter name RuBisCO, is an enzyme (EC 4.1.1.39) that is used in theCalvin cycle to catalyze the first major step of carbon fixation, aprocess by which the atoms of atmospheric carbon dioxide are madeavailable to organisms in the form of energy-rich molecules such assucrose. RuBisCO catalyzes either the carboxylation or the oxygenationof ribulose-1,5-bisphosphate (also known as RuBP) with carbon dioxide oroxygen.

RuBisCO is one of the most abundant proteins on Earth. Accordingly, anumber of homologs and variants of RuBisCO have been identified andgenerated. RuBisCo usually consists of two types of protein subunit,called the large chain (L, about 55,000 Da) and the small chain (S,about 13,000 Da). The enzymatically active substrate (ribulose1,5-bisphosphate) binding sites are located in the large chains thatform dimers in which amino acids from each large chain contribute to thebinding sites. A total of eight large-chain dimers and eight smallchains assemble into a larger complex of about 540,000 Da. In someproteobacteria and dinoflagellates, enzymes consisting of only largesubunits have been found.

Magnesium ions (Mg²⁺) are needed for enzymatic activity. Correctpositioning of Mg²⁺ in the active site of the enzyme involves additionof an “activating” carbon dioxide molecule (CO₂) to a lysine in theactive site (forming a carbamate). Formation of the carbamate is favoredby an alkaline pH. The pH and the concentration of magnesium ions in thefluid compartment (in plants, the stroma of the chloroplast) increasesin the light.

During carbon fixation, the substrate molecules for RuBisCO are ribulose1,5-bisphosphate, carbon dioxide and water. RuBisCO can also allow areaction to occur with molecular oxygen (O₂) instead of carbon dioxide(CO₂).

When carbon dioxide is the substrate, the product of the carboxylasereaction is a highly unstable six-carbon phosphorylated intermediateknown as 3-keto-2-carboxyarabinitol 1,5-bisphosphate, which decays intotwo molecules of glycerate 3-phosphate. The 3-phosphoglycerate can beused to produce larger molecules such as glucose. When molecular oxygenis the substrate, the products of the oxygenase reaction arephosphoglycolate and 3-phosphoglycerate. Phosphoglycolate initiates asequence of reactions called photorespiration, which involves enzymesand cytochromes located in the mitochondria and peroxisomes. In thisprocess, two molecules of phosphoglycolate are converted to one moleculeof carbon dioxide and one molecule of 3-phosphoglycerate, which canreenter the Calvin cycle. Some of the phosphoglycolate entering thispathway can be retained by plants to produce other molecules such asglycine. Some plants, many algae, and photosynthetic bacteria haveovercome this limitation by devising means to increase the concentrationof carbon dioxide around the enzyme, including C4 carbon fixation,crassulacean acid metabolism and using pyrenoid.

RuBisCO is usually active only during the day because ribulose1,5-bisphosphate is not being produced in the dark, due to theregulation of several other enzymes in the Calvin cycle. In addition,the activity of RuBisCO is coordinated with that of the other enzymes ofthe Calvin cycle.

In plants and some algae, another enzyme, RuBisCO activase is used inthe formation of the carbamate in the active site of RuBisCO. Ribulose1,5-bisphosphate (RuBP) substrate binds more strongly to the activesites lacking the carbamate and markedly slows down the “activation”process. In the light, RuBisCO activase promotes the release of theinhibitory RuBP from the catalytic sites. CA1P binds tightly to theactive site of carbamoylated RuBisCO and inhibits catalytic activity. Inthe light, RuBisCO activase also promotes the release of CA1P from thecatalytic sites. After the CA1P is released from RuBisCO, it is rapidlyconverted to a non-inhibitory form by a light-activatedCA1P-phosphatase.

The removal of the inhibitory RuBP, CA1P, and the other inhibitorysubstrate analogs by activase requires the consumption of ATP. Thisreaction is inhibited by the presence of ADP, and, thus, activaseactivity depends on the ratio of these compounds in the chloroplaststroma. Furthermore, in most plants, the sensitivity of activase to theratio of ATP/ADP is modified by the stromal reduction/oxidation (redox)state through another small regulatory protein, thioredoxin. In thismanner, the activity of activase and the activation state of RuBisCO canbe modulated in response to light intensity and, thus, the rate offormation of the ribulose 1,5-bisphosphate substrate.

In cyanobacteria, inorganic phosphate (P_(i)) participates in thecoordinated regulation of photosynthesis. P_(i) binds to the RuBisCOactive site and to another site on the large chain where it caninfluence transitions between activated and less active conformations ofthe enzyme. Activation of bacterial RuBisCO might be particularlysensitive to P_(i) levels which can act in the same way as RuBisCOactivase in higher plants.

The disclosure provides, in some embodiments, recombinant microorganismsthat utilize upregulated RuBisCO to promote carbon fixation and alcoholproduction in photosynthetic organism as described herein, whilecomprising a recombinant non-light engineered redox pathway for NADPHproduction and utilization.

FIG. 1A shows a CO₂ fixation pathway to produce pyruvate via the CBBcycle. FIG. 1B shows a general pathway for production of isobutanol frompyruvate in a recombinant microorganism. FIG. 1C shows pathways for theproduction of various keto acids from pyruvate. Exemplary metabolitesinclude glucose, pyruvate, 1-propanol, isobutanol, 1-butanol, 2-methyl1-butanol, 3-methyl 1-butanol or 2-phenylethanol, and 2-keto acids. Asdepicted in FIG. 1C, exemplary 2-keto acids include 2-ketobutyrate,2-ketoisovalerate, 2-ketovalerate, 2-keto 3-methylvalerate, 2-keto4-methyl-pentanoate and phenylpyruvate. The exemplary 2-keto acids shownin FIG. 1C may be used as metabolic intermediates in the production of1-propanol, isobutanol, 1-butanol, 2-methyl 1-butanol, 3-methyl1-butanol or 2-phenylethanol. For example, as shown in FIG. 1C arecombinant microorganism metabolically engineered to provide elevatedexpression of enzymes encoded by LeuABCD produces 2-ketovalerate from2-ketobutyrate. The 2-ketovalerate metabolite may be used to produce1-butanol by additional enzymes produced by the metabolically modifiedmicroorganism. Additionally, 1-propanol and 2-methyl 1-butanol can beproduced from 2-ketobutyrate and 2-keto-3-methyl-valerate by arecombinant microorganism metabolically engineered to express orover-express enzymes encoded by ilvIHDC, KDC and ADH genes. Further, themetabolite 2-ketoisovalerate can be produced by a recombinantmicroorganism metabolically engineered to express or over-expressenzymes encoded by ilvIHCD genes. This metabolite can then be used inthe production of isobutanol or 3-methyl 1-butanol. The metabolitespyruvate and phenylpyruvate can be used to produce 2-phenylethanol by arecombinant microorganism metabolically engineered to express orover-express enzymes encoded by KDC and ADH. Additional metabolites andgenes are shown in FIG. 1C.

In various embodiments the metabolically engineered microorganisms orcombination cultures provided herein include biochemical pathways forthe production of higher alcohols including isobutanol, 1-butanol,1-propanol, 2-methyl-1-butanol, 3-methyl-1-butanol and 2-phenylethanolfrom a suitable substrate. In various embodiments a recombinantmicroorganism provided herein includes the elevated expression orexpression of a heterologous polypeptide of at least one target enzymeas compared to a parental microorganism. The recombinant microorganismalso produces at least one metabolite involved in a biosynthetic pathwayfor the production of isobutanol, 1-butanol, 1-propanol,2-methyl-1-butanol, 3-methyl-1-butanol or 2-phenylethanol. In general,the microorganisms or combination culture provided herein include atleast one recombinant metabolic pathway that includes a target enzyme.The pathway acts to modify a substrate or metabolic intermediate in theproduction of isobutanol, 1-butanol, 1-propanol, 2-methyl-1-butanol,3-methyl-1-butanol or 2-phenylethanol. The target enzyme is encoded by,and expressed from, a nucleic acid sequence derived from a suitablebiological source. In some embodiments the polynucleotide is a genederived from a bacterial or yeast source.

As used herein, the term “metabolically engineered” or “metabolicengineering” involves rational pathway design and assembly ofbiosynthetic genes, genes associated with operons, and control elementsof such nucleic acid sequences, for the production of a desiredmetabolite, such as a 2-keto acid or high alcohol, in a microorganism.“Metabolically engineered” can further include optimization of metabolicflux by regulation and optimization of transcription, translation,protein stability and protein functionality using genetic engineeringand appropriate culture condition. The biosynthetic genes can beheterologous to the host (e.g., microorganism), either by virtue ofbeing foreign to the host, or being modified by mutagenesis,recombination, and/or association with a heterologous expression controlsequence in an endogenous host cell. Appropriate culture conditions areconditions of culture medium pH, ionic strength, nutritive content,etc.; temperature; oxygen/CO₂/nitrogen content; humidity; and otherculture conditions that permit production of the compound by the hostmicroorganism, i.e., by the metabolic action of the microorganism.Appropriate culture conditions are well known for microorganisms thatcan serve as host cells.

Accordingly, metabolically “engineered” or “modified” microorganisms areproduced via the introduction of genetic material into a host orparental microorganism of choice thereby modifying or altering thecellular physiology and biochemistry of the microorganism. Through theintroduction of genetic material the parental microorganism acquires newproperties, e.g. the ability to produce a new, or greater quantities of,an intracellular metabolite. In an illustrative embodiment, theintroduction of genetic material into a parental microorganism resultsin a new or modified ability to produce an alcohol such as 1-propanol,isobutanol, 1-butanol, 2-methyl 1-butanol, 3-methyl 1-butanol or2-phenylethanol. The genetic material introduced into the parentalmicroorganism contains gene(s), or parts of genes, coding for one ormore of the enzymes involved in a biosynthetic pathway for theproduction of an alcohol and may also include additional elements forthe expression and/or regulation of expression of these genes, e.g.promoter sequences.

Microorganisms provided herein are modified to produce metabolites inquantities not available in the parental microorganism. A “metabolite”refers to any substance produced by metabolism or a substance necessaryfor or taking part in a particular metabolic process. A metabolite canbe an organic compound that is a starting material (e.g., glucose orpyruvate) in production of an intermediate (e.g., 2-keto acid) or inproduction of an end product (e.g., 1-propanol, isobutanol, 1-butanol,2-methyl 1-butanol, 3-methyl 1-butanol or 2-phenylethanol) ofmetabolism. Metabolites can be used to construct more complex molecules,or they can be broken down into simpler ones. Intermediate metabolitesmay be synthesized from other metabolites used, for example, to makemore complex substances, or broken down into simpler compounds, oftenwith the release of chemical energy. End products of metabolism are thefinal result of the breakdown of other metabolites.

The term “biosynthetic pathway”, also referred to as “metabolicpathway”, refers to a set of anabolic or catabolic biochemical reactionsfor converting (transmuting) one chemical species into another. Geneproducts belong to the same “metabolic pathway” if they, in parallel orin series, act on the same substrate, produce the same product, or acton or produce a metabolic intermediate (i.e., metabolite) between thesame substrate and metabolite end product.

The term “substrate” or “suitable substrate” refers to any substance orcompound that is converted or meant to be converted into anothercompound by the action of an enzyme. The term includes not only a singlecompound, but also combinations of compounds, such as solutions,mixtures and other materials which contain at least one substrate, orderivatives thereof. Further, the term “substrate” encompasses not onlycompounds that provide a carbon source suitable for use as a startingmaterial, such as any biomass derived sugar, but also intermediate andend product metabolites used in a pathway associated with ametabolically engineered microorganism as described herein. A “biomassderived sugar” includes, but is not limited to, molecules such asglucose, mannose, xylose, and arabinose or sugars or intermediatesproduced by a photosynthetic microorganism. The term biomass derivedsugar encompasses suitable carbon substrates ordinarily used bymicroorganisms, such as 6 carbon sugars, including but not limited toglucose, lactose, sorbose, fructose, idose, galactose and mannose all ineither D or L form, or a combination of 6 carbon sugars, such as glucoseand fructose, and/or 6 carbon sugar acids including, but not limited to,2-keto-L-gulonic acid, idonic acid (IA), gluconic acid (GA),6-phosphogluconate, 2-keto-D-gluconic acid (2 KDG), 5-keto-D-gluconicacid, 2-ketogluconatephosphate, 2,5-diketo-L-gulonic acid,2,3-L-diketogulonic acid, dehydroascorbic acid, erythorbic acid (EA) andD-mannonic acid.

The term “alcohol” includes for example 1-propanol, isobutanol,1-butanol, 2-methyl 1-butanol, 3-methyl 1-butanol or 2-phenylethanol.The term “1-butanol” generally refers to a straight chain isomer withthe alcohol functional group at the terminal carbon. The straight chainisomer with the alcohol at an internal carbon is sec-butanol or2-butanol. The branched isomer with the alcohol at a terminal carbon isisobutanol, and the branched isomer with the alcohol at the internalcarbon is tert-butanol.

Accordingly, provided herein are recombinant microorganisms that produceisobutanol and in some embodiments may include the elevated expressionof target enzymes such as acetohydroxy acid synthase (ilvIH operon),acetohydroxy acid isomeroreductase (ilvC), dihydroxy-acid dehydratase(ilvD), 2-keto-acid decarboxylase (PDC6, ARO10, THI3, kivd, or pdc),RuBisCo, furmate dehydrogenase and/or a hydrogenase, and alcoholdehydrogenase (ADH2). The microorganism may further include the deletionor inhibition of expression of an adh (e.g., an adhE), ldh (e.g., anldhA), frd (e.g., an frdB, an frdC or an frdBC), fnr, pflB, or pta gene,or any combination thereof, to increase the availability of pyruvate. Insome embodiments the recombinant microorganism may include the elevatedexpression of acetolactate synthase (alsS), acteohydroxy acidisomeroreductase (ilvC), dihydroxy-acid dehydratase (ilvD), 2-keto aciddecarboxylase (PDC6, ARO10, TH13, kivd, or pdc), and alcoholdehydrogenase (ADH2). In one embodiment, the recombinant microorganismis an autophototroph or may be a non-photosynthetic organismrecombinantly engineered to produce the alcohol that is cultured incombination with a autophototroph to fix CO₂. In another embodiment, therecombinant microorganism is a photosynthetic microorganism comprising adecoupled light and dark reaction, wherein the dark reaction comprises arecombinant pathway that utilizes H₂ or formate as a reducing agent. Inone embodiment, the microorganism comprise a heterologous hydrogenaseand/or formate dehydrogenase. In another embodiment, the recombinantmicroorganism comprises a recombinant pathway that utilizes H₂ orformate as a reducing agent. In one embodiment, the microorganismcomprise a heterologous hydrogenase and/or formate dehydrogenase.

Also provided are recombinant microorganisms that produce 1-butanol andmay include the elevated expression of target enzymes such as2-isopropylmalate synthase (leuA), beta-isopropylmalate dehydrogenase(leuB), isopropylmalate isomerase (leuCD operon), threonine dehydratase(ilvA). The microorganism may be a autophotroph microorganism or anon-photosynthetic or heterotrophic microorganism. The microorganism mayfurther include decreased levels of 2-ketoisovalerate,2-keto-3-methyl-valerate, or 2-keto-4-methyl-pentanoate, or anycombination thereof, as compared to a parental microorganism. Inaddition, the microorganism may include the deletion or inhibition ofexpression of an ilvD gene, as compared to a parental microorganism. Arecombinant microorganism that produces 1-butanol and may includefurther elevated expression or activity of pyruvate carboxylase,aspartate aminotransferase, homoserine dehydrogenase,aspartate-semialdehyde dehydrogenase, homoserine kinase, threoninesynthase, L-serine dehydratase, and/or threonine dehydratase, encoded bya nucleic acid sequences derived from the ppc, pyc, aspC, thrA, asd,thrB, thrC, sdaAB, and tdcB genes, respectively. In another embodiment,the recombinant microorganism is a photosynthetic microorganismcomprising a decoupled light and dark reaction, wherein the darkreaction comprises a recombinant pathway that utilizes H₂ or formate asa reducing agent. In one embodiment, the microorganism comprise aheterologous hydrogenase and/or formate dehydrogenase. In anotherembodiment, the recombinant microorganism comprises a recombinantpathway that utilizes H₂ or formate as a reducing agent. In oneembodiment, the microorganism comprise a heterologous hydrogenase and/orformate dehydrogenase.

Also provided are recombinant microorganisms that produce 1-propanol andmay include the elevated expression of target enzymes such asalpha-isopropylmalate synthase (cimA), beta-isopropylmalatedehydrogenase (leuB), isopropylmalate isomerase (leuCD operon) andthreonine dehydratase. In another embodiment, the recombinantmicroorganism is a photosynthetic microorganism comprising a decoupledlight and dark reaction, wherein the dark reaction comprises arecombinant pathway that utilizes H₂ or formate as a reducing agent. Inone embodiment, the microorganism comprise a heterologous hydrogenaseand/or formate dehydrogenase. In another embodiment, the recombinantmicroorganism comprises a recombinant pathway that utilizes H₂ orformate as a reducing agent. In one embodiment, the microorganismcomprise a heterologous hydrogenase and/or formate dehydrogenase.

Also provided are recombinant microorganisms that produce 2-methyl1-butanol and may include the elevated expression of target enzymes suchas threonine dehydratase (ilvA or tdcB), acetohydroxy acid synthase(ilvIH operon), acetohydroxy acid isomeroreductase (ilvC),dihydroxy-acid dehydratase (ilvD), 2-keto-acid decarboxylase (PDC6,ARO10, THI3, kivd, and/or pdc, and alcohol dehydrogenase (ADH2). Inanother embodiment, the recombinant microorganism is a photosyntheticmicroorganism comprising a decoupled light and dark reaction, whereinthe dark reaction comprises a recombinant pathway that utilizes H₂ orformate as a reducing agent. In one embodiment, the microorganismcomprise a heterologous hydrogenase and/or formate dehydrogenase. Inanother embodiment, the recombinant microorganism comprises arecombinant pathway that utilizes H₂ or formate as a reducing agent. Inone embodiment, the microorganism comprise a heterologous hydrogenaseand/or formate dehydrogenase.

Also provided are recombinant photoautotroph microorganism(s) or culturecomprising a photoautotroph and a recombinant non-photosynthetic orphotoheterotroph microorganism that produce 3-methyl 1-butanol and mayinclude the elevated expression of target enzymes such as acetolactatesynthase (alsS), acetohydroxy acid synthase (ilvIH), acetolactatesynthase (ilvMG) or (ilvNB), acetohydroxy acid isomeroreductase (ilvC),dihydroxy-acid dehydratase (ilvD), 2-isopropylmalate synthase (leuA),isopropylmalate isomerase (leuCD operon), beta-isopropylmalatedehydrogenase (leuB), 2-keto-acid decarboxylase (kivd, PDC6, or THI3),and alcohol dehydrogenase (ADH2). In another embodiment, the recombinantmicroorganism is a photosynthetic microorganism comprising a decoupledlight and dark reaction, wherein the dark reaction comprises arecombinant pathway that utilizes H₂ or formate as a reducing agent. Inone embodiment, the microorganism comprise a heterologous hydrogenaseand/or formate dehydrogenase. In another embodiment, the recombinantmicroorganism comprises a recombinant pathway that utilizes H₂ orformate as a reducing agent. In one embodiment, the microorganismcomprise a heterologous hydrogenase and/or formate dehydrogenase.

Also provided are recombinant photoautotroph microorganism(s) or culturecomprising a photoautotroph and a recombinant non-photosynthetic orphotoheterotroph microorganism that produce phenylethanol and mayinclude the elevated expression of target enzymes such as chorismatemutase P/prephenate dehydratase (pheA), chorismate mutase T/prephenatedehydrogenase (tyrA), 2-keto-acid decarboxylase (kivd, PDC6, or THI3),and alcohol dehydrogenase (ADH2). In another embodiment, the recombinantmicroorganism is a photosynthetic microorganism comprising a decoupledlight and dark reaction, wherein the dark reaction comprises arecombinant pathway that utilizes H₂ or formate as a reducing agent. Inone embodiment, the microorganism comprise a heterologous hydrogenaseand/or formate dehydrogenase. In another embodiment, the recombinantmicroorganism comprises a recombinant pathway that utilizes H₂ orformate as a reducing agent. In one embodiment, the microorganismcomprise a heterologous hydrogenase and/or formate dehydrogenase.

As previously noted the target enzymes described throughout thisdisclosure generally produce metabolites. For example, the enzymes2-isopropylmalate synthase (leuA), beta-isopropylmalate dehydrogenase(leuB), and isopropylmalate isomerase (leuCD operon) may produce2-ketovalerate from a substrate that includes 2-ketobutyrate. Inaddition, the target enzymes described throughout this disclosure areencoded by nucleic acid sequences. For example, threonine dehydratasecan be encoded by a nucleic acid sequence derived from an ilvA gene.Acetohydroxy acid synthase can be encoded by a nucleic acid sequencederived from an ilvIH operon. Acetohydroxy acid isomeroreductase can beencoded by a nucleic acid sequence derived from an ilvC gene.Dihydroxy-acid dehydratase can be encoded by a nucleic acid sequencederived from an ilvD gene. 2-keto-acid decarboxylase can be encoded by anucleic acid sequence derived from a PDC6, ARO10, THI3, kivd, and/or pdcgene. Alcohol dehydrogenase can be encoded by a nucleic acid sequencederived from an ADH2 gene. Additional enzymes and exemplary genes aredescribed throughout this document. Homologs of the various polypeptidesand nucleic acid sequences can be derived from any biologic source thatprovides a suitable nucleic acid sequence encoding a suitable enzyme.

It is understood that a range of microorganisms can be modified toinclude a recombinant metabolic pathway suitable for the production ofe.g., 1-propanol, isobutanol, 1-butanol, 2-methyl 1-butanol, 3-methyl1-butanol or 2-phenylethanol. It is also understood that variousmicroorganisms can act as “sources” for genetic material encoding targetenzymes suitable for use in a recombinant microorganism provided herein.The term “microorganism” includes prokaryotic and eukaryoticphotosynthetic microbial species and non-photosynthetic species. Theterms “microbial cells” and “microbes” are used interchangeably with theterm microorganism.

“Bacteria”, or “eubacteria”, refers to a domain of prokaryoticorganisms. Bacteria include at least 11 distinct groups as follows: (1)Gram-positive (gram+) bacteria, of which there are two majorsubdivisions: (1) high G+C group (Actinomycetes, Mycobacteria,Micrococcus, others) (2) low G+C group (Bacillus, Clostridia,Lactobacillus, Staphylococci, Streptococci, Mycoplasmas); (2)Proteobacteria, e.g., Purple photosynthetic+non-photosyntheticGram-negative bacteria (includes most “common” Gram-negative bacteria);(3) Cyanobacteria, e.g., oxygenic phototrophs; (4) Spirochetes andrelated species; (5) Planctomyces; (6) Bacteroides, Flavobacteria; (7)Chlamydia; (8) Green sulfur bacteria; (9) Green non-sulfur bacteria(also anaerobic phototrophs); (10) Radioresistant micrococci andrelatives; (11) Thermotoga and Thermosipho thermophiles.

“Gram-negative bacteria” include cocci, nonenteric rods, and entericrods. The genera of Gram-negative bacteria include, for example,Neisseria, Spirillum, Pasteurella, Brucella, Yersinia, Francisella,Haemophilus, Bordetella, Escherichia, Salmonella, Shigella, Klebsiella,Proteus, Vibrio, Pseudomonas, Bacteroides, Acetobacter, Aerobacter,Agrobacterium, Azotobacter, Spirilla, Serratia, Vibrio, Rhizobium,Chlamydia, Ralstonia, Rickettsia, Treponema, and Fusobacterium.

“Gram positive bacteria” include cocci, nonsporulating rods, andsporulating rods. The genera of gram positive bacteria include, forexample, Actinomyces, Bacillus, Clostridium, Corynebacterium,Erysipelothrix, Lactobacillus, Listeria, Mycobacterium, Myxococcus,Nocardia, Staphylococcus, Streptococcus, and Streptomyces.

Photoautotrophic bacteria are typically Gram-negative rods which obtaintheir energy from sunlight through the processes of photosynthesis. Inthis process, sunlight energy is used in the synthesis of carbohydrates,which in recombinant photoautotrophs can be further used asintermediates in the synthesis of biofuels. In other embodiment, thephotoautotrophs serve as a source of carbohydrates for use bynon-photosynthetic microorganism (e.g., recombinant E. coli) to producebiofuels by a metabolically engineered microorganism. Certainphotoautotrophs called anoxygenic photoautotrophs grow only underanaerobic conditions and neither use water as a source of hydrogen norproduce oxygen from photosynthesis. Other photoautotrophic bacteria areoxygenic photoautotrophs. These bacteria are typically cyanobacteria.They use chlorophyll pigments and photosynthesis in photosyntheticprocesses resembling those in algae and complex plants. During theprocess, they use water as a source of hydrogen and produce oxygen as aproduct of photosynthesis.

Cyanobacteria include various types of bacterial rods and cocci, as wellas certain filamentous forms. The cells contain thylakoids, which arecytoplasmic, platelike membranes containing chlorophyll. The organismsproduce heterocysts, which are specialized cells believed to function inthe fixation of nitrogen compounds.

The term “recombinant microorganism” and “recombinant host cell” areused interchangeably herein and refer to microorganisms that have beengenetically modified to express or over-express endogenous nucleic acidsequences, or to express non-endogenous sequences, such as thoseincluded in a vector. The nucleic acid sequence generally encodes atarget enzyme involved in a metabolic pathway for producing a desiredmetabolite as described above. Accordingly, recombinant microorganismsdescribed herein have been genetically engineered to express orover-express target enzymes not previously expressed or over-expressedby a parental microorganism. It is understood that the terms“recombinant microorganism” and “recombinant host cell” refer not onlyto the particular recombinant microorganism but to the progeny orpotential progeny of such a microorganism.

A “parental microorganism” refers to a cell used to generate arecombinant microorganism. The term “parental microorganism” describes acell that occurs in nature, i.e. a “wild-type” cell that has not beengenetically modified. The term “parental microorganism” also describes acell that has been genetically modified but which does not express orover-express a target enzyme e.g., an enzyme involved in thebiosynthetic pathway for the production of a desired metabolite such as1-propanol, isobutanol, 1-butanol, 2-methyl 1-butanol, 3-methyl1-butanol or 2-phenylethanol. For example, a wild-type microorganism canbe genetically modified to express or over express a first target enzymesuch as thiolase. This microorganism can act as a parental microorganismin the generation of a microorganism modified to express or over-expressa second target enzyme e.g., hydroxybutyryl CoA dehydrogenase. In turn,the microorganism modified to express or over express e.g., thiolase andhydroxybutyryl CoA dehydrogenase can be modified to express or overexpress a third target enzyme e.g., crotonase. Accordingly, a parentalmicroorganism functions as a reference cell for successive geneticmodification events. Each modification event can be accomplished byintroducing a nucleic acid molecule in to the reference cell. Theintroduction facilitates the expression or over-expression of a targetenzyme. It is understood that the term “facilitates” encompasses theactivation of endogenous nucleic acid sequences encoding a target enzymethrough genetic modification of e.g., a promoter sequence in a parentalmicroorganism. It is further understood that the term “facilitates”encompasses the introduction of exogenous nucleic acid sequencesencoding a target enzyme in to a parental microorganism.

In another embodiment a method of producing a recombinant microorganismthat converts a suitable carbon substrate (including CO₂) to e.g.,1-propanol, isobutanol, 1-butanol, 2-methyl 1-butanol, 3-methyl1-butanol or 2-phenylethanol is provided. The method includestransforming a microorganism with one or more recombinant nucleic acidsequences encoding polypeptides that include e.g., a hydrogenase and/ora formate dehydrogenase, acetohydroxy acid synthase operon),acetohydroxy acid isomeroreductase (ilvC), dihydroxy-acid dehydratase(ilvD), 2-keto-acid decarboxylase (PDC6, ARO10, THI3, kivd, or pdc),2-isopropylmalate synthase (leuA), beta-isopropylmalate dehydrogenase(leuB), isopropylmalate isomerase (leuCD operon), threonine dehydratase(ilvA), alpha-isopropylmalate synthase (cimA), beta-isopropylmalatedehydrogenase (leuB), isopropylmalate isomerase (leuCD operon),threonine dehydratase (ilvA), acetolactate synthase (ilvMG or ilvNB),acetohydroxy acid isomeroreductase (ilvC), dihydroxy-acid dehydratase(ilvD), beta-isopropylmalate dehydrogenase (leuB), chorismate mutaseP/prephenate dehydratase (pheA), chorismate mutase T/prephenatedehydrogenase (tyrA), 2-keto-acid decarboxylase (kivd, PDC6, or THI3),and alcohol dehydrogenase activity. Nucleic acid sequences that encodeenzymes useful for generating metabolites including homologs, variants,fragments, related fusion proteins, or functional equivalents thereof,are used in recombinant nucleic acid molecules that direct theexpression of such polypeptides in appropriate host cells, such asbacterial or yeast cells. It is understood that the addition ofsequences which do not alter the encoded activity of a nucleic acidmolecule, such as the addition of a non-functional or non-codingsequence, is a conservative variation of the basic nucleic acid. The“activity” of an enzyme is a measure of its ability to catalyze areaction resulting in a metabolite, i.e., to “function”, and may beexpressed as the rate at which the metabolite of the reaction isproduced. For example, enzyme activity can be represented as the amountof metabolite produced per unit of time or per unit of enzyme (e.g.,concentration or weight), or in terms of affinity or dissociationconstants.

A “protein” or “polypeptide”, which terms are used interchangeablyherein, comprises one or more chains of chemical building blocks calledamino acids that are linked together by chemical bonds called peptidebonds. An “enzyme” means any substance, composed wholly or largely ofprotein, that catalyzes or promotes, more or less specifically, one ormore chemical or biochemical reactions. The term “enzyme” can also referto a catalytic polynucleotide (e.g., RNA or DNA). A “native” or“wild-type” protein, enzyme, polynucleotide, gene, or cell, means aprotein, enzyme, polynucleotide, gene, or cell that occurs in nature.

Accordingly, homologs of enzymes useful for generating metabolites(e.g., keto thiolase, acetyl-CoA acetyltransferase, hydroxybutyryl CoAdehydrogenase, crotonase, crotonyl-CoA reductase, butyryl-coAdehydrogenase, alcohol dehydrogenase (ADH)) are encompassed by themicroorganisms and methods provided herein. The term “homologs” usedwith respect to an original enzyme or gene of a first family or speciesrefers to distinct enzymes or genes of a second family or species whichare determined by functional, structural or genomic analyses to be anenzyme or gene of the second family or species which corresponds to theoriginal enzyme or gene of the first family or species. Most often,homologs will have functional, structural or genomic similarities.Techniques are known by which homologs of an enzyme or gene can readilybe cloned using genetic probes and PCR. Identity of cloned sequences ashomolog can be confirmed using functional assays and/or by genomicmapping of the genes.

A protein has “homology” or is “homologous” to a second protein if thenucleic acid sequence that encodes the protein has a similar sequence tothe nucleic acid sequence that encodes the second protein.Alternatively, a protein has homology to a second protein if the twoproteins have “similar” amino acid sequences. (Thus, the term“homologous proteins” is defined to mean that the two proteins havesimilar amino acid sequences).

As used herein, two proteins (or a region of the proteins) aresubstantially homologous when the amino acid sequences have at leastabout 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% identity. To determine the percent identity of twoamino acid sequences, or of two nucleic acid sequences, the sequencesare aligned for optimal comparison purposes (e.g., gaps can beintroduced in one or both of a first and a second amino acid or nucleicacid sequence for optimal alignment and non-homologous sequences can bedisregarded for comparison purposes). In one embodiment, the length of areference sequence aligned for comparison purposes is at least 30%,typically at least 40%, more typically at least 50%, even more typicallyat least 60%, and even more typically at least 70%, 80%, 90%, 100% ofthe length of the reference sequence. The amino acid residues ornucleotides at corresponding amino acid positions or nucleotidepositions are then compared. When a position in the first sequence isoccupied by the same amino acid residue or nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position (as used herein amino acid or nucleic acid“identity” is equivalent to amino acid or nucleic acid “homology”). Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences, taking into account thenumber of gaps, and the length of each gap, which need to be introducedfor optimal alignment of the two sequences.

When “homologous” is used in reference to proteins or peptides, it isrecognized that residue positions that are not identical often differ byconservative amino acid substitutions. A “conservative amino acidsubstitution” is one in which an amino acid residue is substituted byanother amino acid residue having a side chain (R group) with similarchemical properties (e.g., charge or hydrophobicity). In general, aconservative amino acid substitution will not substantially change thefunctional properties of a protein. In cases where two or more aminoacid sequences differ from each other by conservative substitutions, thepercent sequence identity or degree of homology may be adjusted upwardsto correct for the conservative nature of the substitution. Means formaking this adjustment are well known to those of skill in the art (see,e.g., Pearson et al., 1994, hereby incorporated herein by reference).

The following six groups each contain amino acids that are conservativesubstitutions for one another: 1) Serine (S), Threonine (T); 2) AsparticAcid (D), Glutamic Acid (E); 3) Asparagine (N), Glutamine (Q); 4)Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine(M), Alanine (A), Valine (V), and 6) Phenylalanine (F), Tyrosine (Y),Tryptophan (W).

Sequence homology for polypeptides, which is also referred to as percentsequence identity, is typically measured using sequence analysissoftware. See, e.g., the Sequence Analysis Software Package of theGenetics Computer Group (GCG), University of Wisconsin BiotechnologyCenter, 910 University Avenue, Madison, Wis. 53705. Protein analysissoftware matches similar sequences using measure of homology assigned tovarious substitutions, deletions and other modifications, includingconservative amino acid substitutions. For instance, GCG containsprograms such as “Gap” and “Bestfit” which can be used with defaultparameters to determine sequence homology or sequence identity betweenclosely related polypeptides, such as homologous polypeptides fromdifferent species of organisms or between a wild type protein and amutein thereof. See, e.g., GCG Version 6.1.

A typical algorithm when comparing a inhibitory molecule sequence to adatabase containing a large number of sequences from different organismsis the computer program BLAST (Altschul, 1990; Gish, 1993; Madden, 1996;Altschul, 1997; Zhang, 1997), especially blastp or tblastn (Altschul,1997). Typical parameters for BLASTp are: Expectation value: 10(default); Filter: seg (default); Cost to open a gap: 11 (default); Costto extend a gap: 1 (default); Max. alignments: 100 (default); Word size:11 (default); No. of descriptions: 100 (default); Penalty Matrix:BLOWSUM62.

When searching a database containing sequences from a large number ofdifferent organisms, it is typical to compare amino acid sequences.Database searching using amino acid sequences can be measured byalgorithms other than blastp known in the art. For instance, polypeptidesequences can be compared using FASTA, a program in GCG Version 6.1.FASTA provides alignments and percent sequence identity of the regionsof the best overlap between the query and search sequences (Pearson,1990, hereby incorporated herein by reference). For example, percentsequence identity between amino acid sequences can be determined usingFASTA with its default parameters (a word size of 2 and the PAM250scoring matrix), as provided in GCG Version 6.1, hereby incorporatedherein by reference.

It is understood that the nucleic acid sequences described above include“genes” and that the nucleic acid molecules described above include“vectors” or “plasmids.” For example, a nucleic acid sequence encoding aketo thiolase can be encoded by an atoB gene or homolog thereof, or anfadA gene or homolog thereof. Accordingly, the term “gene”, also calleda “structural gene” refers to a nucleic acid sequence that codes for aparticular sequence of amino acids, which comprise all or part of one ormore proteins or enzymes, and may include regulatory (non-transcribed)DNA sequences, such as promoter sequences, which determine for examplethe conditions under which the gene is expressed. The transcribed regionof the gene may include untranslated regions, including introns,5′-untranslated region (UTR), and 3′-UTR, as well as the codingsequence. The term “nucleic acid” or “recombinant nucleic acid” refersto polynucleotides such as deoxyribonucleic acid (DNA), and, whereappropriate, ribonucleic acid (RNA). The term “expression” with respectto a gene sequence refers to transcription of the gene and, asappropriate, translation of the resulting mRNA transcript to a protein.Thus, as will be clear from the context, expression of a protein resultsfrom transcription and translation of the open reading frame sequence.

The term “operon” refers two or more genes which are transcribed as asingle transcriptional unit from a common promoter. In some embodiments,the genes comprising the operon are contiguous genes. It is understoodthat transcription of an entire operon can be modified (i.e., increased,decreased, or eliminated) by modifying the common promoter.Alternatively, any gene or combination of genes in an operon can bemodified to alter the function or activity of the encoded polypeptide.The modification can result in an increase in the activity of theencoded polypeptide. Further, the modification can impart new activitieson the encoded polypeptide. Exemplary new activities include the use ofalternative substrates and/or the ability to function in alternativeenvironmental conditions.

A “vector” is any means by which a nucleic acid can be propagated and/ortransferred between organisms, cells, or cellular components. Vectorsinclude viruses, bacteriophage, pro-viruses, plasmids, phagemids,transposons, and artificial chromosomes such as YACs (yeast artificialchromosomes), BACs (bacterial artificial chromosomes), and PLACs (plantartificial chromosomes), and the like, that are “episomes,” that is,that replicate autonomously or can integrate into a chromosome of a hostcell. A vector can also be a naked RNA polynucleotide, a naked DNApolynucleotide, a polynucleotide composed of both DNA and RNA within thesame strand, a poly-lysine-conjugated DNA or RNA, a peptide-conjugatedDNA or RNA, a liposome-conjugated DNA, or the like, that are notepisomal in nature, or it can be an organism which comprises one or moreof the above polynucleotide constructs such as an agrobacterium or abacterium.

“Transformation” refers to the process by which a vector is introducedinto a host cell. Transformation (or transduction, or transfection), canbe achieved by any one of a number of means including electroporation,microinjection, biolistics (or particle bombardment-mediated delivery),or agrobacterium mediated transformation.

Those of skill in the art will recognize that, due to the degeneratenature of the genetic code, a variety of DNA compounds differing intheir nucleotide sequences can be used to encode a given amino acidsequence of the disclosure. The native DNA sequence encoding thebiosynthetic enzymes described above are referenced herein merely toillustrate an embodiment of the disclosure, and the disclosure includesDNA compounds of any sequence that encode the amino acid sequences ofthe polypeptides and proteins of the enzymes utilized in the methods ofthe disclosure. In similar fashion, a polypeptide can typically tolerateone or more amino acid substitutions, deletions, and insertions in itsamino acid sequence without loss or significant loss of a desiredactivity. The disclosure includes such polypeptides with alternate aminoacid sequences, and the amino acid sequences encoded by the DNAsequences shown herein merely illustrate embodiments of the disclosure.

The disclosure provides nucleic acid molecules in the form ofrecombinant DNA expression vectors or plasmids, as described in moredetail below, that encode one or more target enzymes. Generally, suchvectors can either replicate in the cytoplasm of the host microorganismor integrate into the chromosomal DNA of the host microorganism. Ineither case, the vector can be a stable vector (i.e., the vector remainspresent over many cell divisions, even if only with selective pressure)or a transient vector (i.e., the vector is gradually lost by hostmicroorganisms with increasing numbers of cell divisions). Thedisclosure provides DNA molecules in isolated (i.e., not pure, butexisting in a preparation in an abundance and/or concentration not foundin nature) and purified (i.e., substantially free of contaminatingmaterials or substantially free of materials with which thecorresponding DNA would be found in nature) forms.

Provided herein are methods for the heterologous expression of one ormore of the biosynthetic genes involved in 1-propanol, isobutanol,1-butanol, 2-methyl 1-butanol, 3-methyl 1-butanol, and/or2-phenylethanol biosynthesis and recombinant DNA expression vectorsuseful in the method. Thus, included within the scope of the disclosureare recombinant expression vectors that include such nucleic acids. Theterm expression vector refers to a nucleic acid that can be introducedinto a host microorganism or cell-free transcription and translationsystem. An expression vector can be maintained permanently ortransiently in a microorganism, whether as part of the chromosomal orother DNA in the microorganism or in any cellular compartment, such as areplicating vector in the cytoplasm. An expression vector also comprisesa promoter that drives expression of an RNA, which typically istranslated into a polypeptide in the microorganism or cell extract. Forefficient translation of RNA into protein, the expression vector alsotypically contains a ribosome-binding site sequence positioned upstreamof the start codon of the coding sequence of the gene to be expressed.Other elements, such as enhancers, secretion signal sequences,transcription termination sequences, and one or more marker genes bywhich host microorganisms containing the vector can be identified and/orselected, may also be present in an expression vector. Selectablemarkers, i.e., genes that confer antibiotic resistance or sensitivity,are used and confer a selectable phenotype on transformed cells when thecells are grown in an appropriate selective medium.

The various components of an expression vector can vary widely,depending on the intended use of the vector and the host cell(s) inwhich the vector is intended to replicate or drive expression.Expression vector components suitable for the expression of genes andmaintenance of vectors in E. coli, yeast, Streptomyces, and othercommonly used cells are widely known and commercially available. Forexample, suitable promoters for inclusion in the expression vectors ofthe disclosure include those that function in eukaryotic or prokaryotichost microorganisms. Promoters can comprise regulatory sequences thatallow for regulation of expression relative to the growth of the hostmicroorganism or that cause the expression of a gene to be turned on oroff in response to a chemical or physical stimulus. For E. coli andcertain other bacterial host cells, promoters derived from genes forbiosynthetic enzymes, antibiotic-resistance conferring enzymes, andphage proteins can be used and include, for example, the galactose,lactose (lac), maltose, tryptophan (trp), beta-lactamase (bla),bacteriophage lambda PL, and T5 promoters. In addition, syntheticpromoters, such as the tac promoter (U.S. Pat. No. 4,551,433) can alsobe used. For E. coli expression vectors, it is useful to include an E.coli origin of replication, such as from pUC, p1P, p1, and pBR.

Thus, recombinant expression vectors contain at least one expressionsystem, which, in turn, is composed of at least a portion of PKS and/orother biosynthetic gene coding sequences operably linked to a promoterand optionally termination sequences that operate to effect expressionof the coding sequence in compatible host cells. The host cells aremodified by transformation with the recombinant DNA expression vectorsof the disclosure to contain the expression system sequences either asextrachromosomal elements or integrated into the chromosome.

Due to the inherent degeneracy of the genetic code, other nucleic acidsequences which encode substantially the same or a functionallyequivalent amino acid sequence can also be used to clone and express thepolynucleotides encoding such enzymes. As previously noted, the term“host cell” is used interchangeably with the term “recombinantmicroorganism” and includes any cell type which is suitable forproducing e.g., 1-propanol, isobutanol, 1-butanol, 2-methyl 1-butanol,3-methyl 1-butanol and/or 2-phenylethanol and susceptible totransformation with a nucleic acid construct such as a vector orplasmid.

As will be understood by those of skill in the art, it can beadvantageous to modify a coding sequence to enhance its expression in aparticular host. The genetic code is redundant with 64 possible codons,but most organisms typically use a subset of these codons. The codonsthat are utilized most often in a species are called optimal codons, andthose not utilized very often are classified as rare or low-usagecodons. Codons can be substituted to reflect the preferred codon usageof the host, a process sometimes called “codon optimization” or“controlling for species codon bias.”

Optimized coding sequences containing codons preferred by a particularprokaryotic or eukaryotic host (see also, Murray et al. (1989) Nucl.Acids Res. 17:477-508) can be prepared, for example, to increase therate of translation or to produce recombinant RNA transcripts havingdesirable properties, such as a longer half-life, as compared withtranscripts produced from a non-optimized sequence. Translation stopcodons can also be modified to reflect host preference. For example,typical stop codons for S. cerevisiae and mammals are UAA and UGA,respectively. The typical stop codon for monocotyledonous plants is UGA,whereas insects and E. coli commonly use UAA as the stop codon (Dalphinet al. (1996) Nucl. Acids Res. 24: 216-218). Methodology for optimizinga nucleotide sequence for expression in a plant is provided, forexample, in U.S. Pat. No. 6,015,891, and the references cited therein.

A nucleic acid of the disclosure can be amplified using cDNA, mRNA oralternatively, genomic DNA, as a template and appropriateoligonucleotide primers according to standard PCR amplificationtechniques and those procedures described in the Examples section below.The nucleic acid so amplified can be cloned into an appropriate vectorand characterized by DNA sequence analysis. Furthermore,oligonucleotides corresponding to nucleotide sequences can be preparedby standard synthetic techniques, e.g., using an automated DNAsynthesizer.

It is also understood that an isolated nucleic acid molecule encoding apolypeptide homologous to the enzymes described herein can be created byintroducing one or more nucleotide substitutions, additions or deletionsinto the nucleotide sequence encoding the particular polypeptide, suchthat one or more amino acid substitutions, additions or deletions areintroduced into the encoded protein. Mutations can be introduced intothe nucleic acid sequence by standard techniques, such as site-directedmutagenesis and PCR-mediated mutagenesis. In contrast to those positionswhere it may be desirable to make a non-conservative amino acidsubstitutions (see above), in some positions it is preferable to makeconservative amino acid substitutions. A “conservative amino acidsubstitution” is one in which the amino acid residue is replaced with anamino acid residue having a similar side chain. Families of amino acidresidues having similar side chains have been defined in the art. Thesefamilies include amino acids with basic side chains (e.g., lysine,arginine, histidine), acidic side chains (e.g., aspartic acid, glutamicacid), uncharged polar side chains (e.g., glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine).

In another embodiment a method for producing e.g., 1-propanol,isobutanol, 1-butanol, 2-methyl 1-butanol, 3-methyl 1-butanol or2-phenylethanol is provided. The method includes culturing a recombinantphotoautotroph microorganism(s) or culture comprising a photoautotrophand a recombinant non-photosynthetic or photoheterotroph microorganismas provided herein in the presence of a suitable substrate (e.g., CO₂)and under conditions suitable for the conversion of the substrate to1-propanol, isobutanol, 1-butanol, 2-methyl 1-butanol, 3-methyl1-butanol or 2-phenylethanol. The alcohol produced by a microorganism orculture provided herein can be detected by any method known to theskilled artisan. Culture conditions suitable for the growth andmaintenance of a recombinant microorganism provided herein are describedin the Examples below. The skilled artisan will recognize that suchconditions can be modified to accommodate the requirements of eachmicroorganism.

The disclosure provides accession numbers for various genes, homologsand variants useful in the generation of recombinant microorganismdescribed herein. It is to be understood that homologs and variantsdescribed herein are exemplary and non-limiting. Additional homologs,variants and sequences are available to those of skill in the art usingvarious databases including, for example, the National Center forBiotechnology Information (NCBI) access to which is available on theWorld-Wide-Web.

Several thousand Ribulose-1,5-bisphosphate carbxylase/oxygenase andother CO₂ fixation enzymes are known and their sequences are readilyavailable in the art using various search criteria and web-sites. Forexample, the methods and compositions of the disclosure may utilizeRibulose-1,5-bisphosphate carboxylase/oxygenase (RubisCo)—smallsubunit—cbbS, Ribulose-1,5-bisphosphate carbyxlase/oxygenase(RubisCo)—large subunit cbbL, Rubisco activase, rbcL, rbcS and variantsand homologs thereof in the disclosure. In yet other relatedembodiments, the engineered can further comprise engineered rbcL nucleicacid, engineered rbcS nucleic acid, and engineered phosphoribulokinase.Rubisco polypeptides of the useful in the disclosure include Rubiscolarge subunit polypeptides (“rbcL”), Rubisco small subunit polypeptides(“rbcS”), and Rubisco large/small polypeptides (“rbcLS”). Large andsmall subunits may be combined in different combinations with each othertogether in a single enzyme having Rubisco specific activity.Alternatively, the large and small subunits of the may be combined withthe large and small subunits from a wild type Rubisco polypeptides toform a polypeptide having Rubisco activity. Exemplaryribulose-1,5-bisphosophate carboxylase/oxygenases include spinach form IRubisco Spinacia oleracea; gi:7636117; CAB88737, Archaeoglobus fulgidusDSM 4304 rbcL-1 (gi:2648975; AAB86661); Sinorhizobium meliloti 1021(gi:15140252; CAC48779); Mesorhizobium loti MAFF303099 (gi:14026595;BAB53192); Chlorobium limicola f. thiosulfatophilum (gi:13173182;AAK14332); C. tepidum TLS (gi:21647784; AAM72993); R. palustris(gi:78490428; ZP_(—)00842677); R. palustris (gi:77687805;ZP_(—)00802991); R. rubrum (gi:48764419; ZP_(—)00268971); Bordetellabronchiseptica RB50 (gi:33567621; CAE31534); Burkholderia fungorum LB400(gi:48788861; ZP_(—)00284840); B. clausii KSM-K16 (gi:56909783;BAD64310); Bacillus thuringiensis serovar konkukian strain 97-27(gi:49333072; AAT63718); Geobacillus kaustophilus HTA426 (gi:56379330;BAD75238); Bacillus licheniformis ATCC14580 (gi:52003120; AAU23062);Bacillus anthracis strain A2012 (gi:65321428; ZP_(—)00394387); Bacilluscereus E33L (gi:51974924; AAU16474); B. subtilis subsp. subtilis strain168 (gi:2633730; CAB 13232). Accession numbers are from GenBank andsequences associated with those accession numbers are incorporatedherein by reference. In addition, variants comprising RuBisCo activityand having at least 85%, 90%, 95%, 98%, 99% identity to any of theforegoing sequences is also encompassed by the disclosure.

Ethanol Dehydrogenase (also referred to as Aldehyde-alcoholdehydrogenase) is encoded in E. coli by adhE. adhE comprises threeactivities: alcohol dehydrogenase (ADH); acetaldehyde/acetyl-CoAdehydrogenase (ACDH); pyruvate-formate-lyase deactivase (PFLdeactivase); PFL deactivase activity catalyzes the quenching of thepyruvate-formate-lyase catalyst in an iron, NAD, and CoA dependentreaction. Homologs are known in the art (see, e.g., aldehyde-alcoholdehydrogenase (Polytomella sp. Pringsheim 198.80)gi|40644910|emb|CAD42653.2|(40644910); aldehyde-alcohol dehydrogenase(Clostridium botulinum A str. ATCC 3502)gi|148378348|ref|YP_(—)001252889.1|(148378348); aldehyde-alcoholdehydrogenase (Yersinia pestis CO92)gi|16122410|ref|NP_(—)405723.1|(16122410); aldehyde-alcoholdehydrogenase (Yersinia pseudotuberculosis IP 32953)gi|51596429|ref|YP_(—)070620.1|(51596429); aldehyde-alcoholdehydrogenase (Yersinia pestis C092)gi|115347889|emb|CAL20810.1|(115347889); aldehyde-alcohol dehydrogenase(Yersinia pseudotuberculosis IP 32953)gi|51589711|emb|CAH21341.1|(51589711); Aldehyde-alcohol dehydrogenase(Escherichia coli CFT073)gi|26107972|gb|AAN80172.1|AE016760_(—)3|(26107972); aldehyde-alcoholdehydrogenase (Yersinia pestis biovar Microtus str. 91001)gi|45441777|ref|NP_(—)993316.1|(45441777); aldehyde-alcoholdehydrogenase (Yersinia pestis biovar Microtus str. 91001)gi|45436639|gb|AAS62193.1|(45436639); aldehyde-alcohol dehydrogenase(Clostridium perfringens ATCC 13124)gi|10798574|ref|YP_(—)697219.1|(110798574); aldehyde-alcoholdehydrogenase (Shewanella oneidensisMR-1)gi|24373696|ref|NP_(—)717739.1|(24373696); aldehyde-alcoholdehydrogenase (Clostridium botulinum A str. ATCC 19397)gi|153932445|ref|YP_(—)001382747.1|(153932445); aldehyde-alcoholdehydrogenase (Yersinia pestis biovar Antigua str. E1979001)gi|165991833|gb|EDR44134.1|(165991833); aldehyde-alcohol dehydrogenase(Clostridium botulinum A str. Hall)gi|153937530|ref|YP_(—)001386298.1|(153937530); aldehyde-alcoholdehydrogenase (Clostridium perfringens ATCC 13124)gi|110673221|gb|ABG82208.1|(110673221); aldehyde-alcohol dehydrogenase(Clostridium botulinum A str. Hall)gi|152933444|gb|ABS38943.1|(152933444); aldehyde-alcohol dehydrogenase(Yersinia pestis biovar Orientalis str. F1991016)gi|165920640|gb|EDR37888.1|(165920640); aldehyde-alcohol dehydrogenase(Yersinia pestis biovar Orientalis str.IP275)gi|165913933|gb|EDR32551.1|(165913933); aldehyde-alcoholdehydrogenase (Yersinia pestis Angola)gi|162419116|ref|YP_(—)001606617.1|(162419116); aldehyde-alcoholdehydrogenase (Clostridium botulinum F str. Langeland)gi|153940830|ref|YP_(—)001389712.1|(153940830); aldehyde-alcoholdehydrogenase (Escherichia coli HS)gi|157160746|ref|YP_(—)001458064.1|(157160746); aldehyde-alcoholdehydrogenase (Escherichia coli E24377A)gi|157155679|ref|YP_(—)001462491.1|(157155679); aldehyde-alcoholdehydrogenase (Yersinia enterocolitica subsp. enterocolitica 8081)gi|123442494|ref|YP_(—)001006472.1|(123442494); aldehyde-alcoholdehydrogenase (Synechococcus sp. JA-3-3Ab)gi|86605191|ref|YP_(—)473954.1|(86605191); aldehyde-alcoholdehydrogenase (Listeria monocytogenes str. 4b F2365)gi|46907864|ref|YP_(—)014253.1|(46907864); aldehyde-alcoholdehydrogenase (Enterococcus faecalis V583)gi|29375484|ref|NP_(—)814638.1|(29375484); aldehyde-alcoholdehydrogenase (Streptococcus agalactiae 2603V/R)gi|22536238|ref|NP_(—)687089.1|(22536238); aldehyde-alcoholdehydrogenase (Clostridium botulinum A str. ATCC 19397)gi|152928489|gb|ABS33989.1|(152928489); aldehyde-alcohol dehydrogenase(Escherichia coli E24377A) gi|157077709|gb|ABV17417.1|(157077709);aldehyde-alcohol dehydrogenase (Escherichia coli HS)gi|157066426|gb|ABV05681.1|(157066426); aldehyde-alcohol dehydrogenase(Clostridium botulinum F str. Langeland)gi|152936726|gb|ABS42224.1|(152936726); aldehyde-alcohol dehydrogenase(Yersinia pestis CA88-4125) gi|149292312|gb|EDM42386.1|(149292312);aldehyde-alcohol dehydrogenase (Yersinia enterocolitica subsp.enterocolitica 8081) gi|122089455|emb|CAL12303.1|(122089455);aldehyde-alcohol dehydrogenase (Chlamydomonas reinhardtii)gi|92084840|emb|CAF04128.1|(92084840); aldehyde-alcohol dehydrogenase(Synechococcus sp. JA-3-3Ab) gi|86553733|gb|ABC98691.1|(86553733);aldehyde-alcohol dehydrogenase (Shewanella oneidensis MR-1)gi|24348056|gb|AAN55183.1|AE015655_(—)9(24348056); aldehyde-alcoholdehydrogenase (Enterococcus faecalis V583)gi|29342944|gb|AAO80708.1|(29342944); aldehyde-alcohol dehydrogenase(Listeria monocytogenes str. 4b F2365)gi|46881133|gb|AAT04430.1|(46881133); aldehyde-alcohol dehydrogenase(Listeria monocytogenes str. 1/2a F6854)gi|47097587|ref|ZP_(—)00235115.1|(47097587); aldehyde-alcoholdehydrogenase (Listeria monocytogenes str. 4b H7858)gi|47094265|ref|ZP_(—)00231973.1|(47094265); aldehyde-alcoholdehydrogenase (Listeria monocytogenes str. 4b H7858)gi|47017355|gb|EAL08180.1|(47017355); aldehyde-alcohol dehydrogenase(Listeria monocytogenes str. 1/2a F6854)gi|47014034|gb|EAL05039.1|(47014034); aldehyde-alcohol dehydrogenase(Streptococcus agalactiae 2603V/R)gi|22533058|gb|AAM98961.1|AE014194_(—)6(22533058)p; aldehyde-alcoholdehydrogenase (Yersinia pestis biovar Antigua str. E1979001)gi|166009278|ref|ZP_(—)02230176.1|(166009278); aldehyde-alcoholdehydrogenase (Yersinia pestis biovar Orientalis str. IP275)gi|165938272|ref|ZP_(—)02226831.1|(165938272); aldehyde-alcoholdehydrogenase (Yersinia pestis biovar Orientalis str. F1991016)gi|165927374|ref|ZP_(—)02223206.1|(165927374); aldehyde-alcoholdehydrogenase (Yersinia pestis Angola)gi|162351931|gb|ABX85879.1|(162351931); aldehyde-alcohol dehydrogenase(Yersinia pseudotuberculosis IP 31758)gi|153949366|ref|YP_(—)001400938.1|(153949366); aldehyde-alcoholdehydrogenase (Yersinia pseudotuberculosis IP 31758)gi|152960861|gb|ABS48322.1|(152960861); aldehyde-alcohol dehydrogenase(Yersinia pestis CA88-4125)gi|149365899|ref|ZP_(—)01887934.1|(149365899); Acetaldehydedehydrogenase (acetylating) (Escherichia coli CFT073)gi|26247570|ref|NP_(—)753610.1|(26247570); aldehyde-alcoholdehydrogenase (includes: alcohol dehydrogenase; acetaldehydedehydrogenase (acetylating) (EC 1.2.1.10) (acdh); pyruvate-formate-lyasedeactivase (pfl deactivase)) (Clostridium botulinum A str. ATCC 3502)gi|148287832|emb|CAL81898.1|(148287832); aldehyde-alcohol dehydrogenase(Includes: Alcohol dehydrogenase (ADH); Acetaldehyde dehydrogenase(acetylating) (ACDH); Pyruvate-formate-lyase deactivase (PFLdeactivase)) gi|71152980|sp|P0A9Q7.2|ADHE_ECOLI(71152980);aldehyde-alcohol dehydrogenase (includes: alcohol dehydrogenase andacetaldehyde dehydrogenase, and pyruvate-formate-lyase deactivase(Erwinia carotovora subsp. atroseptica SCR11043)gi|50121254|ref|YP_(—)050421.1|(50121254); aldehyde-alcoholdehydrogenase (includes: alcohol dehydrogenase and acetaldehydedehydrogenase, and pyruvate-formate-lyase deactivase (Erwinia carotovorasubsp. atroseptica SCR11043) gi|49611780|emb|CAG75229.1|(49611780);Aldehyde-alcohol dehydrogenase (Includes: Alcohol dehydrogenase (ADH);Acetaldehyde dehydrogenase (acetylating) (ACDH))gi|19858620|sp|P33744.3|ADHE_CLOAB(19858620); Aldehyde-alcoholdehydrogenase (Includes: Alcohol dehydrogenase (ADH); Acetaldehydedehydrogenase (acetylating) (ACDH); Pyruvate-formate-lyase deactivase(PFL deactivase)) gi|71152683|sp|P0A9Q8.2|ADHE_ECO57(71152683);aldehyde-alcohol dehydrogenase (includes: alcohol dehydrogenase;acetaldehyde dehydrogenase (acetylating); pyruvate-formate-lyasedeactivase (Clostridium difficile 630)gi|126697906|ref|YP_(—)001086803.1|(126697906); aldehyde-alcoholdehydrogenase (includes: alcohol dehydrogenase; acetaldehydedehydrogenase (acetylating); pyruvate-formate-lyase deactivase(Clostridium difficile 630) gi|115249343|emb|CAJ67156.1|(115249343);Aldehyde-alcohol dehydrogenase (includes: alcohol dehydrogenase (ADH)and acetaldehyde dehydrogenase (acetylating) (ACDH);pyruvate-formate-lyase deactivase (PFL deactivase)) (Photorhabdusluminescens subsp. laumondii TTO1)gi|37526388|ref|NP_(—)929732.1|(37526388); aldehyde-alcoholdehydrogenase 2 (includes: alcohol dehydrogenase; acetaldehydedehydrogenase) (Streptococcus pyogenes str. Manfredo)gi|134271169|emb|CAM29381.1|(134271169); Aldehyde-alcohol dehydrogenase(includes: alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase(acetylating) (ACDH); pyruvate-formate-lyase deactivase (PFLdeactivase)) (Photorhabdus luminescens subsp. laumondii TTO1)gi|136785819|emb|CAE14870.1|(36785819); aldehyde-alcohol dehydrogenase(includes: alcohol dehydrogenase and pyruvate-formate-lyase deactivase(Clostridium difficile 630)gi|126700586|ref|YP_(—)001089483.1|(126700586); aldehyde-alcoholdehydrogenase (includes: alcohol dehydrogenase andpyruvate-formate-lyase deactivase (Clostridium difficile 630)gi|115252023|emb|CAJ69859.1|(115252023); aldehyde-alcohol dehydrogenase2 (Streptococcus pyogenes str. Manfredo)gi|139472923|ref|YP_(—)001127638.1|(139472923); aldehyde-alcoholdehydrogenase E (Clostridium perfringens str. 13)gi|18311513|ref|NP_(—)563447.1|(18311513); aldehyde-alcoholdehydrogenase E (Clostridium perfringens str. 13)gi|18146197|dbj|BAB82237.1|(18146197); Aldehyde-alcohol dehydrogenase,ADHE1 (Clostridium acetobutylicum ATCC 824)gi|15004739|ref|NP_(—)149199.1|(15004739); Aldehyde-alcoholdehydrogenase, ADHE1 (Clostridium acetobutylicum ATCC 824)gi|14994351|gb|AAK76781.1|AE001438_(—)34(14994351); Aldehyde-alcoholdehydrogenase 2 (Includes: Alcohol dehydrogenase (ADH);acetaldehyde/acetyl-CoA dehydrogenase (ACDH))gi|2492737|sp|Q24803.1|ADH2_ENTHI(2492737); alcohol dehydrogenase(Salmonella enterica subsp. enterica serovar Typhi str. CT18)gi|16760134|ref|NP_(—)455751.1|(16760134); and alcohol dehydrogenase(Salmonella enterica subsp. enterica serovar Typhi)gi|16502428|emb|CAD08384.1|(16502428)), each sequence associated withthe accession number is incorporated herein by reference in itsentirety.

Lactate Dehydrogenase (also referred to as D-lactate dehydrogenase andfermentive dehydrognase) is encoded in E. coli by ldhA and catalyzes theNADH-dependent conversion of pyruvate to D-lactate. ldhA homologs andvariants are known. In fact there are currently 1664 bacterial lactatedehydrogenases available through NCBI. For example, such homologs andvariants include, for example, D-lactate dehydrogenase (D-LDH)(Fermentative lactate dehydrogenase)gi|1730102|sp|P52643.1|LDHD_ECOLI(1730102); D-lactate dehydrogenasegi|1049265|gb|AAB51772.1|(1049265); D-lactate dehydrogenase (Escherichiacoli APEC O1) gi|117623655|ref|YP_(—)852568.1|(117623655); D-lactatedehydrogenase (Escherichia coli CFT073)gi|26247689|ref|NP_(—)753729.1|(26247689); D-lactate dehydrogenase(Escherichia coli O157:H7 EDL933)gi|15801748|ref|NP_(—)287766.1|(15801748); D-lactate dehydrogenase(Escherichia coli APEC O1) gi|115512779|gb|ABJ00854.1|(115512779);D-lactate dehydrogenase (Escherichia coli CFT073)gi|26108091|gb|AAN80291.1|AE016760_(—)150(26108091); fermentativeD-lactate dehydrogenase, NAD-dependent (Escherichia coli K12)gi|16129341|ref|NP_(—)415898.1|(16129341); fermentative D-lactatedehydrogenase, NAD-dependent (Escherichia coli UTI89)gi|91210646|ref|IP_(—)540632.1|(91210646); fermentative D-lactatedehydrogenase, NAD-dependent (Escherichia coli K12)gi|1787645|gb|AAC74462.1|(1787645); fermentative D-lactatedehydrogenase, NAD-dependent (Escherichia coli W3110)gi|89108227|ref|AP_(—)002007.1|(89108227); fermentative D-lactatedehydrogenase, NAD-dependent (Escherichia coli W3110)gi|1742259|dbj|BAA14990.1|(1742259); fermentative D-lactatedehydrogenase, NAD-dependent (Escherichia coli UTI89)gi|91072220|gb|ABE07101.1|(91072220); fermentative D-lactatedehydrogenase, NAD-dependent (Escherichia coli O157:H7 EDL933)gi|12515320|gb|AAG56380.1|AE005366_(—)6(12515320); fermentativeD-lactate dehydrogenase. (Escherichia coli O157:H7 str. Sakai)gi|13361468|dbj|BAB35425.1|(13361468); COG1052: Lactate dehydrogenaseand related dehydrogenases (Escherichia coli 101-1)gi|83588593|ref|ZP_(—)00927217.1|(83588593); COG1052: Lactatedehydrogenase and related dehydrogenases (Escherichia coli 53638)gi|75515985|ref|ZP_(—)00738103.1|(75515985); COG1052: Lactatedehydrogenase and related dehydrogenases (Escherichia coli E22)gi|75260157|ref|ZP_(—)00731425.1|(75260157); COG1052: Lactatedehydrogenase and related dehydrogenases (Escherichia coli F11)gi|75242656|ref|ZP_(—)00726400.1|(75242656); COG1052: Lactatedehydrogenase and related dehydrogenases (Escherichia coli E110019)gi|75237491|ref|ZP_(—)00721524.1|(75237491); COG1052: Lactatedehydrogenase and related dehydrogenases (Escherichia coli B7A)gi|75231601|ref|ZP_(—)00717959.1|(75231601); and COG1052: Lactatedehydrogenase and related dehydrogenases (Escherichia coli B171)gi|75211308|ref|ZP_(—)00711407.1|(75211308), each sequence associatedwith the accession number is incorporated herein by reference in itsentirety.

Two membrane-bound, FAD-containing enzymes are responsible for thecatalysis of fumarate and succinate interconversion; the fumaratereductase is used in anaerobic growth, and the succinate dehydrogenaseis used in aerobic growth. Fumarate reductase comprises multiplesubunits (e.g., frdA, B, and C in E. coli). Modification of any one ofthe subunits can result in the desired activity herein. For example, aknockout of frdB, frdC or frdBC is useful in the methods of thedisclosure. Frd homologs and variants are known. For example, homologsand variants includes, for example, Fumarate reductase subunit D(Fumarate reductase 13 kDa hydrophobic protein)gi|67463543|sp|P0A8Q3.1|FRDD_ECOLI(67463543); Fumarate reductase subunitC (Fumarate reductase 15 kDa hydrophobic protein)gi|1346037|sp|P20923.2|FRDC_PROVU(1346037); Fumarate reductase subunit D(Fumarate reductase 13 kDa hydrophobic protein)gi|120499|sp|P20924.1|FRDD_PROVU(120499); Fumarate reductase subunit C(Fumarate reductase 15 kDa hydrophobic protein)gi|67463538|sp|P0A8Q0.1|FRDC_ECOLI(67463538); fumarate reductaseiron-sulfur subunit (Escherichia coli) gi|145264|gb|AAA23438.1|(145264);fumarate reductase flavoprotein subunit (Escherichia coli)gi|145263|gb|AAA23437.1|(145263); Fumarate reductase flavoproteinsubunit gi|37538290|sp|P17412.3|FRDA_WOLSU(37538290); Fumarate reductaseflavoprotein subunit gi|120489|sp|P00363.3|FRDA_ECOLI(120489); Fumaratereductase flavoprotein subunit gi|120490|sp|P20922.1|FRDA_PROVU(120490);Fumarate reductase flavoprotein subunit precursor (Flavocytochrome c)(Flavocytochrome c3) (Fcc3)gi|119370087|sp|Q07WU7.2|FRDA_SHEFN(119370087); Fumarate reductaseiron-sulfur subunit gi|81175308|sp|P0AC47.2|FRDB_ECOLI(81175308);Fumarate reductase flavoprotein subunit (Flavocytochrome c)(Flavocytochrome c3) (Fcc3)gi|119370088|sp|P0C278.1|FRDA_SHEFR(119370088); Frd operonuncharacterized protein C gi|140663|sp|P20927.1|YFRC_PROVU(140663); Frdoperon probable iron-sulfur subunit Agi|140661|sp|P20925.1|YFRA_PROVU(140661); Fumarate reductase iron-sulfursubunit gi|120493|sp|P20921.2|FRDB_PROVU(120493); Fumarate reductaseflavoprotein subunit gi|2494617|sp|O06913.2|FRDA_HELPY(2494617);Fumarate reductase flavoprotein subunit precursor (Iron(III)-inducedflavocytochrome C3) (Ifc3) gi|13878499|sp|Q9Z4P0.1|FRD2_SHEFN(13878499);Fumarate reductase flavoprotein subunitgi|54041009|sp|P64174.1|FRDA_MYCTU(54041009); Fumarate reductaseflavoprotein subunit gi|54037132|sp|P64175.1|FRDA_MYCBO(54037132);Fumarate reductase flavoprotein subunitgi|12230114|sp|Q9ZMP0.1|FRDA_HELPJ(12230114); Fumarate reductaseflavoprotein subunit gi|1169737|sp|P44894.1|FRDA_HAEIN(1169737);fumarate reductase flavoprotein subunit (Wolinella succinogenes)gi|13160058|emb|CAA04214.2|(13160058); Fumarate reductase flavoproteinsubunit precursor (Flavocytochrome c) (FL cyt)gi|25452947|sp|P83223.2|FRDA_SHEON(25452947); fumarate reductaseiron-sulfur subunit (Wolinella succinogenes)gi|2282000|emb|CAA04215.1|(2282000); and fumarate reductase cytochrome bsubunit (Wolinella succinogenes) gi|2281998|emb|CAA04213.1|(2281998),each sequence associated with the accession number is incorporatedherein by reference in its entirety.

Acetate kinase is encoded in E. coli by ackA. AckA is involved inconversion of acetyl-coA to acetate. Specifically, ackA catalyzes theconversion of acetyl-phosphate to acetate. AckA homologs and variantsare known. The NCBI database list approximately 1450 polypeptides asbacterial acetate kinases. For example, such homologs and variantsinclude acetate kinase (Streptomyces coelicolor A3(2))gi|21223784|ref|NP_(—)629563.1|(21223784); acetate kinase (Streptomycescoelicolor A3(2)) gi|6808417|emb|CAB70654.1|(6808417); acetate kinase(Streptococcus pyogenes M1 GAS)gi|15674332|ref|NP_(—)268506.1|(15674332); acetate kinase (Campylobacterjejuni subsp. jejuni NCTC 11168)gi|15792038|ref|NP_(—)281861.1|(15792038); acetate kinase (Streptococcuspyogenes M1 GAS) gi|13621416|gb|AAK33227.1|(13621416); acetate kinase(Rhodopirellula baltica SH 1) gi|32476009|ref|NP_(—)869003.1|(32476009);acetate kinase (Rhodopirellula baltica SH 1)gi|32472045|ref|NP_(—)865039.1|(32472045); acetate kinase (Campylobacterjejuni subsp. jejuni NCTC 11168)gi|112360034|emb|CAL34826.1|(112360034); acetate kinase (Rhodopirellulabaltica SH 1) gi|32446553|emb|CAD76388.1|(32446553); acetate kinase(Rhodopirellula baltica SH 1) gi|32397417|emb|CAD72723.1|(32397417);AckA (Clostridium kluyveri DSM 555)gi|153954016|ref|YP_(—)001394781.1|(153954016); acetate kinase(Bifidobacterium longum NCC2705)gi|23465540|ref|NP_(—)696143.1|(23465540); AckA (Clostridium kluyveriDSM 555) gi|146346897|gb|EDK33433.1|(146346897); Acetate kinase(Corynebacterium diphtheriae) gi|38200875|emb|CAE50580.1|(38200875);acetate kinase (Bifidobacterium longum NCC2705)gi|23326203|gb|AAN24779.1|(23326203); Acetate kinase (Acetokinase)gi|67462089|sp|P0A6A3.1|ACKA_ECOLI(67462089); and AckA (Bacilluslicheniformis DSM 13) gi|52349315|gb|AAU41949.1|(52349315), thesequences associated with such accession numbers are incorporated hereinby reference.

Phosphate acetyltransferase is encoded in E. coli by pta. PTA isinvolved in conversion of acetate to acetyl-CoA. Specifically, PTAcatalyzes the conversion of acetyl-coA to acetyl-phosphate. PTA homologsand variants are known. There are approximately 1075 bacterial phosphateacetyltransferases available on NCBI. For example, such homologs andvariants include phosphate acetyltransferase Pta (Rickettsia felisURRWXCal2) gi|67004021|gb|AAY60947.1|(67004021); phosphateacetyltransferase (Buchnera aphidicola str. Cc (Cinara cedri))gi|116256910|gb|ABJ90592.1|(116256910); pta (Buchnera aphidicola str. Cc(Cinara cedri)) gi|116515056|ref|YP_(—)802685.1|(116515056); pta(Wigglesworthia glossinidia endosymbiont of Glossina brevipalpis)gi|125166135|dbj|BAC24326.1|(25166135); Pta (Pasteurella multocidasubsp. multocida str. Pm70) gi|12720993|gb|AAK02789.1|(12720993); Pta(Rhodospirillum rubrum) gi|125989720|gb|AAN75024.1|(25989720); pta(Listeria welshimeri serovar 6b str. SLCC5334)gi|116742418|emb|CAK21542.1|(116742418); Pta (Mycobacterium avium subsp.paratuberculosis K-10) gi|41398816|gb|AAS06435.1|(41398816); phosphateacetyltransferase (pta) (Borrelia burgdorferi B31)gi|5594934|ref|NP_(—)212723.1|(15594934); phosphate acetyltransferase(pta) (Borrelia burgdorferi B31) gi|2688508|gb|AAB91518.1|(2688508);phosphate acetyltransferase (pta) (Haemophilus influenzae Rd KW20)gi|1574131|gb|AAC22857.1|(1574131); Phosphate acetyltransferase Pta(Rickettsia bellii RML369-C) gi|91206026|ref|YP_(—)538381.1|(91206026);Phosphate acetyltransferase Pta (Rickettsia bellii RML369-C)gi|91206025|ref|YP_(—)538380.1|(91206025); phosphate acetyltransferasepta (Mycobacterium tuberculosis F11)gi|148720131|gb|ABR04756.1|(148720131); phosphate acetyltransferase pta(Mycobacterium tuberculosis str. Haarlem)gi|134148886|gb|EBA40931.1|(134148886); phosphate acetyltransferase pta(Mycobacterium tuberculosis C) gi|124599819|gb|EAY58829.1|(124599819);Phosphate acetyltransferase Pta (Rickettsia bellii RML369-C)gi|91069570|gb|ABE05292.1|(91069570); Phosphate acetyltransferase Pta(Rickettsia bellii RML369-C) gi|91069569|gb|ABE05291.1|(91069569);phosphate acetyltransferase (pta) (Treponema pallidum subsp. pallidumstr. Nichols) gi|15639088|ref|NP_(—)218534.1|(15639088); and phosphateacetyltransferase (pta) (Treponema pallidum subsp. pallidum str.Nichols) gi|3322356|gb|AAC65090.1|(3322356), each sequence associatedwith the accession number is incorporated herein by reference in itsentirety.

Pyruvate-formate lyase (Formate acetylytransferase) is an enzyme thatcatalyzes the conversion of pyruvate to acetyl)-coA and formate. It isinduced by pfl-activating enzyme under anaerobic conditions bygeneration of an organic free radical and decreases significantly duringphosphate limitation. Formate acetylytransferase is encoded in E. coliby pflB. PFLB homologs and variants are known. For examples, suchhomologs and variants include, for example, Formate acetyltransferase 1(Pyruvate formate-lyase 1) gi|129879|sp|P09373.21 PFLB_ECOLI(129879);formate acetyltransferase 1 (Yersinia pestis C092)gi|16121663|ref|NP_(—)404976.1|(16121663); formate acetyltransferase 1(Yersinia pseudotuberculosis IP 32953)gi|51595748|ref|YP_(—)069939.1|(51595748); formate acetyltransferase 1(Yersinia pestis biovar Microtus str. 91001)gi|45441037|ref|NP_(—)992576.1|(45441037); formate acetyltransferase 1(Yersinia pestis CO92) gi|115347142|emb|CAL20035.1|(115347142); formateacetyltransferase 1 (Yersinia pestis biovar Microtus str. 91001)gi|45435896|gb|AAS61453.1|(45435896); formate acetyltransferase 1(Yersinia pseudotuberculosis IP 32953)gi|51589030|emb|CAH20648.1|(51589030); formate acetyltransferase 1(Salmonella enterica subsp. enterica serovar Typhi str. CT18)gi|16759843|ref|NP_(—)455460.1|(16759843); formate acetyltransferase 1(Salmonella enterica subsp. enterica serovar Paratyphi A str. ATCC 9150)gi|56413977|ref|YP_(—)151052.1|(56413977); formate acetyltransferase 1(Salmonella enterica subsp. enterica serovar Typhi)gi|16502136|emb|CAD05373.1|(16502136); formate acetyltransferase 1(Salmonella enterica subsp. enterica serovar Paratyphi A str. ATCC 9150)gi|56128234|gb|AAV77740.1|(56128234); formate acetyltransferase 1(Shigella dysenteriae Sd197) gi|82777577|ref|YP_(—)403926.1|(82777577);formate acetyltransferase 1 (Shigella flexneri 2a str. 2457T)gi|30062438|ref|NP_(—)836609.1|(30062438); formate acetyltransferase 1(Shigella flexneri 2a str. 2457T) gi|30040684|gb|AAP16415.1|(30040684);formate acetyltransferase 1 (Shigella flexneri 5 str. 8401)gi|110614459|gb|ABF03126.1|(110614459); formate acetyltransferase 1(Shigella dysenteriae Sd197) gi|81241725|gb|ABB62435.1|(81241725);formate acetyltransferase 1 (Escherichia coli O157:H7 EDL933)gi|12514066|gb|AAG55388.1|AE005279_(—)8(12514066); formateacetyltransferase 1 (Yersinia pestis KIM)gi|22126668|ref|NP_(—)670091.1|(22126668); formate acetyltransferase 1(Streptococcus agalactiae A909)gi|176787667|ref|YP_(—)330335.1|(76787667); formate acetyltransferase 1(Yersinia pestis KIM) gi|21959683|gb|AAM86342.1|AE013882_(—)3(21959683);formate acetyltransferase 1 (Streptococcus agalactiae A909)gi|76562724|gb|ABA45308.1|(76562724); formate acetyltransferase 1(Yersinia enterocolitica subsp. enterocolitica 8081)gi|123441844|ref|YP_(—)001005827.1|(123441844); formateacetyltransferase 1 (Shigella flexneri 5 str. 8401)gi|110804911|ref|YP_(—)688431.1|(110804911); formate acetyltransferase 1(Escherichia coli UTI89) gi|191210004|ref|YP_(—)539990.1|(91210004);formate acetyltransferase 1Sb227) (Shigella boydii Sb227)gi|82544641|ref|YP_(—)408588.1|(82544641); formate acetyltransferase 1(Shigella sonnei Ss046) gi|74311459|ref|YP_(—)309878.1|(74311459);formate acetyltransferase 1 (Klebsiella pneumoniae subsp. pneumoniae MGH78578) gi|152969488|ref|YP_(—)001334597.1|(152969488); formateacetyltransferase 1 (Salmonella enterica subsp. enterica serovar TyphiTy2) gi|29142384|ref|NP_(—)805726.1|(29142384) formate acetyltransferase1 (Shigella flexneri 2a str. 301)gi|24112311|ref|NP_(—)706821.1|(24112311); formate acetyltransferase 1(Escherichia coli O157:H7 EDL933)gi|15800764|ref|NP_(—)286778.1|(15800764); formate acetyltransferase 1(Klebsiella pneumoniae subsp. pneumoniae MGH 78578)gi|150954337|gb|ABR76367.1|(150954337); formate acetyltransferase 1(Yersinia pestis CA88-4125)gi|149366640|ref|ZP_(—)01888674.1|(149366640); formate acetyltransferase1 (Yersinia pestis CA88-4125) gi|149291014|gb|EDM41089.1|(149291014);formate acetyltransferase 1 (Yersinia enterocolitica subsp.enterocolitica 8081) gi|122088805|emb|CAL11611.1|(122088805); formateacetyltransferase 1 (Shigella sonnei Ss046)gi|73854936|gb|AAZ87643.1|(73854936); formate acetyltransferase 1(Escherichia coli UT189) gi|91071578|gb|ABE06459.1|(91071578); formateacetyltransferase 1 (Salmonella enterica subsp. enterica serovar TyphiTy2) gi|29138014|gb|AAO69575.1|(29138014); formate acetyltransferase 1(Shigella boydii Sb227) gi|81246052|gb|ABB66760.1|(81246052); formateacetyltransferase 1 (Shigella flexneri 2a str. 301)gi|24051169|gb|AAN42528.1|(24051169); formate acetyltransferase 1(Escherichia coli O157:H7 str. Sakai)gi|13360445|dbj|BAB34409.1|(13360445); formate acetyltransferase 1(Escherichia coli O157:H7 str. Sakai)gi|15830240|ref|NP_(—)309013.1|(15830240); formate acetyltransferase 1(pyruvate formate-lyase 1) (Photorhabdus luminescens subsp. laumondiiTTO1) gi|36784986|emb|CAE13906.1|(36784986); formate. acetyltransferase1 (pyruvate formate-lyase 1) (Photorhabdus luminescens subsp. laumondiiTTO1) gi|37525558|ref|NP_(—)928902.1|(37525558); formateacetyltransferase (Staphylococcus aureus subsp. aureus Mu50)gi|14245993|dbj|BAB56388.1|(14245993); formate acetyltransferase(Staphylococcus aureus subsp. aureus Mu50)gi|15923216|ref|NP_(—)370750.1|(15923216); Formate acetyltransferase(Pyruvate formate-lyase) gi|81706366|sp|Q7A7X6.1|PFLB_STAAN(81706366);Formate acetyltransferase (Pyruvate formate-lyase)gi|81782287|sp|Q99WZ7.1|PFLB_STAAM(81782287); Formate acetyltransferase(Pyruvate formate-lyase) gi|81704726|sp|Q7A1W9.1|PFLB_STAAW(81704726);formate acetyltransferase (Staphylococcus aureus subsp. aureus Mu3)gi|156720691|dbj|BAF77108.1|(156720691); formate acetyltransferase(Erwinia carotovora subsp. atroseptica SCR11043)gi|50121521|ref|YP_(—)050688.1|(50121521); formate acetyltransferase(Erwinia carotovora subsp. atroseptica SCR11043)gi|49612047|emb|CAG75496.1|(49612047); formate acetyltransferase(Staphylococcus aureus subsp. aureus str. Newman)gi|150373174|dbj|BAF66434.1|(150373174); formate acetyltransferase(Shewanella oneidensis MR-1) gi|24374439|ref|NP_(—)718482.1|(24374439);formate acetyltransferase (Shewanella oneidensis MR-1)gi|124349015|gb|AAN55926.1|AE015730_(—)3(24349015); formateacetyltransferase (Actinobacillus pleuropneumoniae serovar 3 str. JL03)gi|165976461|ref|YP_(—)001652054.1|(165976461); formateacetyltransferase (Actinobacillus pleuropneumoniae serovar 3 str. JL03)gi|165876562|gb|ABY69610.1|(165876562); formate acetyltransferase(Staphylococcus aureus subsp. aureus MW2)gi|21203365|dbj|BAB94066.1|(21203365); formate acetyltransferase(Staphylococcus aureus subsp. aureus N315)gi|13700141|dbj|BAB41440.1|(13700141); formate acetyltransferase(Staphylococcus aureus subsp. aureus str. Newman)gi|151220374|ref|YP_(—)001331197.1|(151220374); formateacetyltransferase (Staphylococcus aureus subsp. aureus Mu3)gi|156978556|ref|YP_(—)001440815.1|(156978556); formateacetyltransferase (Synechococcus sp. JA-2-3B′ a(2-13))gi|86607744|ref|YP_(—)476506.1|(86607744); formate acetyltransferase(Synechococcus sp. JA-3-3Ab) gi|86605195|ref|YP_(—)473958.1|(86605195);formate acetyltransferase (Streptococcus pneumoniae D39)gi|116517188|ref|YP_(—)815928.1|(116517188); formate acetyltransferase(Synechococcus sp. JA-2-3B′ a(2-13))gi|86556286|gb|ABD01243.1|(86556286); formate acetyltransferase(Synechococcus sp. JA-3-3Ab) gi|86553737|gb|ABC98695.1|(86553737);formate acetyltransferase (Clostridium novyi NT)gi|118134908|gb|ABK61952.1|(118134908); formate acetyltransferase(Staphylococcus aureus subsp. aureus MRSA252)gi|49482458|ref|YP_(—)039682.1|(49482458); and formate acetyltransferase(Staphylococcus aureus subsp. aureus MRSA252)gi|49240587|emb|CAG39244.1|(49240587), each sequence associated with theaccession number is incorporated herein by reference in its entirety.

Alpha isopropylmalate synthase (EC 2.3.3.13, sometimes referred to a2-isopropylmalate synthase, alpha-IPM synthetase) catalyzes thecondensation of the acetyl group of acetyl-CoA with3-methyl-2-oxobutanoate (2-oxoisovalerate) to form3-carboxy-3-hydroxy-4-methylpentanoate (2-isopropylmalate). Alphaisopropylmalate synthase is encoded in E. coli by leuA. LeuA homologsand variants are known. For example, such homologs and variants include,for example, 2-isopropylmalate synthase (Corynebacterium glutamicum)gi|452382|emb|CAA50295.1|(452382); 2-isopropylmalate synthase(Escherichia coli K12) gi|116128068|ref|NP_(—)414616.1|(16128068);2-isopropylmalate synthase (Escherichia coli K12)gi|1786261|gb|AAC73185.1|(1786261); 2-isopropylmalate synthase(Arabidopsis thaliana) gi|15237194|ref|NP_(—)197692.1|(15237194);2-isopropylmalate synthase (Arabidopsis thaliana)gi|42562149|ref|NP_(—)173285.2|(42562149); 2-isopropylmalate synthase(Arabidopsis thaliana) gi|15221125|ref|NP_(—)177544.1|(15221125);2-isopropylmalate synthase (Streptomyces coelicolor A3(2))gi|32141173|ref|NP_(—)733575.1|(32141173); 2-isopropylmalate synthase(Rhodopirellula baltica SH 1) gi|32477692|ref|NP_(—)870686.1|(32477692);2-isopropylmalate synthase (Rhodopirellula baltica SH 1)gi|32448246|emb|CAD77763.1|(32448246); 2-isopropylmalate synthase(Akkermansia muciniphila ATCC BAA-835)gi|166241432|gb|EDR53404.1|(166241432); 2-isopropylmalate synthase(Herpetosiphon aurantiacus ATCC 23779)gi|159900959|ref|YP_(—)001547206.1|(159900959); 2-isopropylmalatesynthase (Dinoroseobacter shibae DFL 12)gi|159043149|ref|YP_(—)001531943.1|(159043149); 2-isopropylmalatesynthase (Salinispora arenicola CNS-205)gi|159035933|ref|YP_(—)001535186.1|(159035933); 2-isopropylmalatesynthase (Clavibacter michiganensis subsp. michiganensis NCPPB 382)gi|148272757|ref|YP_(—)001222318.1|(148272757); 2-isopropylmalatesynthase (Escherichia coli B)gi|124530643|ref|ZP_(—)01701227.1|(124530643); 2-isopropylmalatesynthase (Escherichia coli C str. ATCC 8739)gi|124499067|gb|EAY46563.1|(124499067); 2-isopropylmalate synthase(Bordetella pertussis Tohama I)gi|33591386|ref|NP_(—)879030.1|(33591386); 2-isopropylmalate synthase(Polynucleobacter necessarius STIR 1)gi|164564063|ref|ZP_(—)02209880.1|(164564063); 2-isopropylmalatesynthase (Polynucleobacter necessarius STIR1)gi|164506789|gb|EDQ94990.1|(164506789); and 2-isopropylmalate synthase(Bacillus weihenstephanensis KBAB4)gi|163939313|ref|YP_(—)001644197.1|(163939313), any sequence associatedwith the accession number is incorporated herein by reference in itsentirety.

BCAA aminotransferases catalyze the formation of branched chain aminoacids (BCAA). A number of such aminotranferases are known and areexemplified by ilvE in E. coli. Exemplary homologs and variants includesequences designated by the following accession numbers: ilvE(Microcystic aeruginosa PCC 7806)gi|159026756|emb|CAO86637.1|(159026756); IlvE (Escherichia coli)gi|87117962|gb|ABD20288.1|(87117962); IlvE (Escherichia coli)gi|87117960|gb|ABD20287.1|(87117960); IlvE (Escherichia coli)gi|87117958|gb|ABD20286.1|(87117958); IlvE (Shigella flexneri)gi|87117956|gb|ABD20285.1|(87117956); IlvE (Shigella flexneri)gi|87117954|gb|ABD20284.1|(87117954); IlvE (Shigella flexneri)gi|87117952|gb|ABD20283.1|(87117952); IlvE (Shigella flexneri)gi|87117950|gb|ABD20282.1|(87117950); IlvE (Shigella flexneri)gi|87117948|gb|ABD20281.1|(87117948); IlvE (Shigella flexneri)gi|87117946|gb|ABD20280.1|(87117946); IlvE (Shigella flexneri)gi|87117944|gb|ABD20279.1|(87117944); IlvE (Shigella flexneri)gi|87117942|gb|ABD20278.1|(87117942); IlvE (Shigella flexneri)gi|87117940|gb|ABD20277.1|(87117940); IlvE (Shigella flexneri)gi|87117938|gb|ABD20276.1|(87117938); IlvE (Shigella dysenteriae)gi|87117936|gb|ABD20275.1|(87117936); IlvE (Shigella dysenteriae)gi|87117934|gb|ABD20274.1|(87117934); IlvE (Shigella dysenteriae)gi|87117932|gb|ABD20273.1|(87117932); IlvE (Shigella dysenteriae)gi|87117930|gb|ABD20272.1|(87117930); and IlvE (Shigella dysenteriae)gi|87117928|gb|ABD20271.1|(87117928), each sequence associated with theaccession number is incorporated herein by reference.

Tyrosine aminotransferases catalyzes transamination for bothdicarboxylic and aromatic amino-acid substrates. A tyrosineaminotransferase of E. coli is encoded by the gene tyrB. TyrB homologsand variants are known. For example, such homologs and variants includetyrB (Bordetella petrii) gi|163857093|ref|YP_(—)001631391.1|(163857093);tyrB (Bordetella petrii) gi|163260821|emb|CAP43123.1|(163260821);aminotransferase gi|1551844|gb|AAA24704.1|(551844); aminotransferase(Bradyrhizobium sp. BTAi1) gi|146404387|gb|ABQ32893.1|(146404387);tyrosine aminotransferase TyrB (Salmonella enterica)gi|4775574|emb|CAB40973.2|(4775574); tyrosine aminotransferase(Salmonella typhimurium LT2) gi|16422806|gb|AAL23072.1|(16422806); andtyrosine aminotransferase gi|148085|gb|AAA24703.1|(148085), eachsequence of which is incorporated herein by reference.

Pyruvate oxidase catalyzes the conversion of pyruvate to acetate andCO₂. In E. coli, pyruvate oxidase is encoded by poxB. PoxB and homologsand variants thereof include, for example, pyruvate oxidase; PoxB(Escherichia coli) gi|685128|gb|AAB31180.1∥bbm|34845|bbs|154716(685128);PoxB (Pseudomonas fluorescens) gi|32815820|gb|AAP88293.1|(32815820);poxB (Escherichia coli) gi|25269169|emb|CAD57486.1|(25269169); pyruvatedehydrogenase (Salmonella enterica subsp. enterica serovar Typhi)gi|16502101|emb|CAD05337.1|(16502101); pyruvate oxidase (Lactobacillusplantarum) gi|41691702|gb|AAS10156.1|(41691702); pyruvate dehydrogenase(Bradyrhizobium japonicum) gi|20257167|gb|AAM12352.1|(20257167);pyruvate dehydrogenase (Yersinia pestis KIM)gi|22126698|ref|NP_(—)670121.1|(22126698); pyruvate dehydrogenase(cytochrome) (Yersinia pestis biovar Antigua str. B42003004)gi|166211240|ref|ZP_(—)02237275.1|(166211240); pyruvate dehydrogenase(cytochrome) (Yersinia pestis biovar Antigua str. B42003004)gi|166207011|gb|EDR51491.1|(166207011); pyruvate dehydrogenase(Pseudomonas syringae pv. tomato str. DC3000)gi|28869703|ref|NP_(—)792322.1|(28869703); pyruvate dehydrogenase(Salmonella typhimurium LT2) gi|16764297|ref|NP_(—)459912.1|(16764297);pyruvate dehydrogenase (Salmonella enterica subsp. enterica serovarTyphi str. CT18) gi|16759808|ref|NP_(—)455425.1|(16759808); pyruvatedehydrogenase (cytochrome) (Coxiella burnetii Dugway 5J108-111)gi|154706110|ref|YP_(—)001424132.1|(154706110); pyruvate dehydrogenase(Clavibacter michiganensis subsp. michiganensis NCPPB 382)gi|148273312|ref|YP_(—)001222873.1|(148273312); pyruvate oxidase(Lactobacillus acidophilus NCFM)gi|58338213|ref|YP_(—)194798.1|(58338213); and pyruvate dehydrogenase(Yersinia pestis C092) gi|16121638|ref|NP_(—)404951.1|(16121638), thesequences of each accession number are incorporated herein by reference.

L-threonine 3-dehydrogenase (EC 1.1.1.103) catalyzes the conversion ofL-threonine to L-2-amino-3-oxobutanoate. The gene tdh encodes anL-threonine 3-dehydrogenase. There are approximately 700 L-threonine3-dehydrogenases from bacterial organism recognized in NCBI. Varioushomologs and variants of tdh include; for example L-threonine3-dehydrogenase gi|135560|sp|P07913.11TDH_ECOLI(135560); L-threonine3-dehydrogenase gi|166227854|sp|A4TSC6.1|TDH_YERPP(166227854);L-threonine 3-dehydrogenasegi|166227853|sp|A1JHX8.1|TDH_YERE8(166227853); L-threonine3-dehydrogenase gi|166227852|sp|A6UBM6.1|TDH_SINMW(166227852);L-threonine 3-dehydrogenasegi|166227851|sp|A1RE07.1|TDH_SHESW(166227851); L-threonine3-dehydrogenase gi|166227850|sp|A0L2Q3.11TDH_SHESA(166227850);L-threonine 3-dehydrogenasegi|166227849|sp|A4YCC5.1|TDH_SHEPC(166227849); L-threonine3-dehydrogenase gi|166227848|sp|A3QJC8.1|TDH_SHELP(166227848);L-threonine 3-dehydrogenasegi|166227847|sp|A6WUG6.1|TDH_SHEB8(166227847); L-threonine3-dehydrogenase gi|166227846|sp|A3CYN0.1|TDH_SHEB5(166227846);L-threonine 3-dehydrogenasegi|166227845|sp|A1S1Q3.11TDH_SHEAM(166227845); L-threonine3-dehydrogenase gi|166227844|sp|A4FND4.11TDH_SACEN(166227844);L-threonine 3-dehydrogenasegi|166227843|sp|A1SVW5.1|TDH_PSYIN(166227843); L-threonine3-dehydrogenase gi|166227842|sp|A51GK7.1|TDH_LEGPC(166227842);L-threonine 3-dehydrogenasegi|166227841|sp|A6TFL2.1|TDH_KLEP7(166227841); L-threonine3-dehydrogenase gi|166227840|sp|A41Z92.1|TDH_FRATW(166227840);L-threonine 3-dehydrogenasegi|166227839|sp|A0Q5K3.1|TDH_FRATN(166227839); L-threonine3-dehydrogenase gi|166227838|sp|A7NDM9.1|TDH_FRATF(166227838);L-threonine 3-dehydrogenasegi|166227837|sp|A7MID0.1|TDH_ENTS8(166227837); and L-threonine3-dehydrogenase gi|166227836|sp|A1AHF3.1|TDH_ECOK1|(166227836), thesequences associated with each accession number are incorporated hereinby reference.

Acetohydroxy acid synthases (e.g. ilvH) and acetolactate synthases(e.g., alsS, ilvB, ilvI) catalyze the synthesis of the branched-chainamino acids (valine, leucine, and isoleucine). IlvH encodes anacetohydroxy acid synthase in E. coli (see, e.g., acetohydroxy acidsynthase AHAS III (IlvH) (Escherichia coli)gi|40846|emb|CAA38855.1|(40846), incorporated herein by reference).Homologs and variants as well as operons comprising ilvH are known andinclude, for example, ilvH (Microcystis aeruginosa PCC7806)gi|159026908|emb|CA089159.1|(159026908); IlvH (Bacillusamyloliquefaciens FZB42) gi|154686966|ref|YP_(—)001422127.1|(154686966);IlvH (Bacillus amyloliquefaciens FZB42)gi|154352817|gb|ABS74896.1|(154352817); IlvH (Xenorhabdus nematophila)gi|131054140|gb|AB032787.1|(131054140); IlvH (Salmonella typhimurium)gi|7631124|gb|AAF65177.1|AF117227_(—)2(7631124), ilvN (Listeria innocua)gi|16414606|emb|CAC97322.1|(16414606); ilvN (Listeria monocytogenes)gi|16411438|emb|CAD00063.1|(16411438); acetohydroxy acid synthase(Caulobacter crescentus) gi|408939|gb|AAA23048.1|(408939); acetohydroxyacid synthase I, small subunit (Salmonella enterica subsp. entericaserovar Typhi) gi|16504830|emb|CAD03199.1|(16504830); acetohydroxy acidsynthase, small subunit (Tropheryma whipplei TW08/27)gi|28572714|ref|NP_(—)789494.1|(28572714); acetohydroxy acid synthase,small subunit (Tropheryma whipplei TW08/27)gi|28410846|emb|CAD67232.1|(28410846); acetohydroxy acid synthase I,small subunit (Salmonella enterica subsp. enterica serovar Paratyphi Astr. ATCC 9150) gi|56129933|gb|AAV79439.1|(56129933); acetohydroxy acidsynthase small subunit; acetohydroxy acid synthase, small subunitgi|551779|gb|AAA62430.1|(551779); acetohydroxy acid synthase I, smallsubunit (Salmonella enterica subsp. enterica serovar Typhi Ty2)gi|29139650|gb|AA071216.1|(29139650); acetohydroxy acid synthase smallsubunit (Streptomyces cinnamonensis)gi|5733116|gb|AAD49432.1|AF175526_(—)1|(5733116); acetohydroxy acidsynthase large subunit; and acetohydroxy acid synthase, large subunitgi|400334|gb|AAA62429.1|(400334), the sequences associated with theaccession numbers are incorporated herein by reference. Acetolactatesynthase genes include aIsS and ilvI. Homologs of ilvI and aIsS areknown and include, for example, acetolactate synthase small subunit(Bifidobacterium longum NCC2705) gi|23325489|gb|AAN24137.1|(23325489);acetolactate synthase small subunit (Geobacillus stearothermophilus)gi|19918933|gb|AAL99357.1|(19918933); acetolactate synthase (Azoarcussp. BH72) gi|119671178|emb|CAL95091.1|(119671178); Acetolactate synthasesmall subunit (Corynebacterium diphtheriae)gi|38199954|emb|CAE49622.1|(38199954); acetolactate synthase (Azoarcussp. BH72) gi|119669739|emb|CAL93652.1|(119669739); acetolactate synthasesmall subunit (Corynebacterium jeikeium K411)gi|68263981|emb|CA137469.1|(68263981); acetolactate synthase smallsubunit (Bacillus subtilis) gi|1770067|emb|CAA99562.1|(1770067);Acetolactate synthase isozyme 1 small subunit (AHAS-I)(Acetohydroxy-acid synthase I small subunit) (ALS-I)gi|83309006|sp|P0ADF8.1|ILVN_ECOLI(83309006); acetolactate synthaselarge subunit (Geobacillus stearothermophilus)gi|19918932|gb|AAL99356.1|(19918932); and Acetolactate synthase, smallsubunit (Thermoanaerobacter tengcongensis MB4)gi|20806556|ref|NP_(—)621727.1|(20806556), the sequences associated withthe accession numbers are incorporated herein by reference. There areapproximately 1120 ilvB homologs and variants listed in NCBI.

Acetohydroxy acid isomeroreductase is the second enzyme in parallelpathways for the biosynthesis of isoleucine and valine. IlvC encodes anacetohydroxy acid isomeroreductase in E. coli. Homologs and variants ofilvC are known and include, for example, acetohydroxyacidreductoisomerase (Schizosaccharomyces pombe 972h-)gi|162312317|ref|NP_(—)001018845.2|(162312317); acetohydroxyacidreductoisomerase (Schizosaccharomyces pombe)gi|3116142|emb|CAA18891.1|(3116142); acetohydroxyacid reductoisomerase(Saccharomyces cerevisiae YJM789)gi|151940879|gb|EDN59261.1|(151940879); Ilv5p: acetohydroxy acidreductoisomerase (Saccharomyces cerevisiae)gi|1609403|gb|AAB67753.1|(609403); ACL198Wp (Ashbya gossypii ATCC 10895)gi|45185490|ref|NP_(—)983206.1|(45185490); ACL198Wp (Ashbya gossypiiATCC 10895) gi|44981208|gb|AAS51030.1|(44981208); acetohydroxy-acidisomeroreductase; Ilv5x (Saccharomyces cerevisiae)gi|957238|gb|AAB33579.1∥bbm|369068|bbs|165406(957238); acetohydroxy-acidisomeroreductase; Ilv5g (Saccharomyces cerevisiae)gi|957236|gb|AAB33578.1∥bbm|369064|bbs|165405(957236); and ketol-acidreductoisomerase (Schizosaccharomyces pombe)gi|2696654|dbj|BAA24000.1|(2696654), each sequence associated with theaccession number is incorporated herein by reference.

Dihydroxy-acid dehydratases catalyzes the fourth step in thebiosynthesis of isoleucine and valine, the dehydratation of2,3-dihydroxy-isovaleic acid into alpha-ketoisovaleric acid. IlvD andilv3 encode a dihydroxy-acid dehydratase. Homologs and variants ofdihydroxy-acid dehydratases are known and include, for example, IlvD(Mycobacterium leprae) gi|2104594|emb|CAB08798.1|(2104594);dihydroxy-acid dehydratase (Tropheryma whipplei TW08/27)gi|28410848|emb|CAD67234.1|(28410848); dihydroxy-acid dehydratase(Mycobacterium leprae) gi|13093837|emb|CAC32140.1|(13093837);dihydroxy-acid dehydratase (Rhodopirellula baltica SH 1)gi|32447871|emb|CAD77389.1|(32447871); and putative dihydroxy-aciddehydratase (Staphylococcus aureus subsp. aureus MRSA252)gi|49242408|emb|CAG41121.1|(49242408), each sequence associated with theaccession numbers are incorporated herein by reference.

2-ketoacid decarboxylases catalyze the conversion of a 2-ketoacid to therespective aldehyde. For example, 2-ketoisovalerate decarboxylasecatalyzes the conversion of 2-ketoisovalerate to isobutyraldehyde. Anumber of 2-ketoacid decarboxylases are known and are exemplified by thepdc, pdc1, pdc5, pdc6, aro10, thl3, kdcA and kivd genes. Exemplaryhomologs and variants useful for the conversion of a 2-ketoacid to therespective aldehyde comprise sequences designated by the followingaccession numbers and identified enzymatic activity:gi|44921617|gb|AAS49166.1| branched-chain alpha-keto acid decarboxylase(Lactococcus lactis); gi|15004729|ref|NP_(—)149189.1| Pyruvatedecarboxylase (Clostridium acetobutylicum ATCC 824);gi|82749898|ref|YP_(—)415639.11 probable pyruvate decarboxylase(Staphylococcus aureus RF122); gi|77961217|ref|ZP_(—)00825060.1|COG3961: Pyruvate decarboxylase and related thiaminepyrophosphate-requiring enzymes (Yersinia mollaretii ATCC 43969);gi|71065418|ref|YP_(—)264145.1| putative pyruvate decarboxylase(Psychrobacter arcticus 273-4); gi|16761331|ref|NP_(—)456948.1| putativedecarboxylase (Salmonella enterica subsp. enterica serovar Typhi str.CT18); gi|93005792|ref|YP_(—)580229.1| Pyruvate decarboxylase(Psychrobacter cryohalolentis K5); gi|23129016|ref|ZP_(—)00110850.1|COG3961: Pyruvate decarboxylase and related thiaminepyrophosphate-requiring enzymes (Nostoc punctiforme PCC 73102);gi|16417060|gb|AAL18557.1|AF354297_(—)1 pyruvate decarboxylase (Sarcinaventriculi); gi|15607993|ref|NP_(—)215368.1|PROBABLE PYRUVATE ORINDOLE-3-PYRUVATE DECARBOXYLASE PDC (Mycobacterium tuberculosis H37Rv);gi|41406881|ref|NP_(—)959717.1|Pdc (Mycobacterium avium subsp.paratuberculosis K-10); gi|91779968|ref|YP_(—)555176.1| putativepyruvate decarboxylase (Burkholderia xenovorans LB400);gi|15828161|ref|NP_(—)302424.1| pyruvate (or indolepyruvate)decarboxylase (Mycobacterium leprae TN);gi|118616174|ref|YP_(—)904506.1| pyruvate or indole-3-pyruvatedecarboxylase Pdc (Mycobacterium ulcerans Agy99);gi|67989660|ref|NP_(—)001018185.1| hypothetical protein SPAC3H8.01(Schizosaccharomyces pombe 972h-);gi|21666011|gb|AAM73540.11AF282847_(—)1 pyruvate decarboxylase PdcB(Rhizopus oryzae); gi|69291130|ref|ZP_(—)00619161.1| Pyruvatedecarboxylase:Pyruvate decarboxylase (Kineococcus radiotoleransSRS30216); gi|66363022|ref|XP_(—)628477.1| pyruvate decarboxylase(Cryptosporidium parvum Iowa II); gi|70981398|ref|XP_(—)731481.1|pyruvate decarboxylase (Aspergillus fumigatus Af293);gi|121704274|ref|XP_(—)001270401.1| pyruvate decarboxylase, putative(Aspergillus clavatus NRRL 1); gi|119467089|ref|XP_(—)001257351.1|pyruvate decarboxylase, putative (Neosartorya fischeri NRRL 181);gi|26554143|ref|NP_(—)758077.1| pyruvate decarboxylase (Mycoplasmapenetrans HF-2); gi|21666009|gb|AAM73539.1|AF282846_(—)1 pyruvatedecarboxylase PdcA (Rhizopus oryzae).

Alcohol dehydrogenases (adh) catalyze the final step of amino acidcatabolism, conversion of an aldehyde to a long chain or complexalcohol. Various adh genes are known in the art. As indicated hereinadh1 homologs and variants include, for example, adh2, adh3, adh4, adh5,adh 6 and sfa1 (see, e.g., SFA (Saccharomyces cerevisiae)gi|1288591|emb|CAA48161.1| (288591); the sequence associated with theaccession number is incorporated herein by reference).

Citramalate synthase catalyzes the condensation of pyruvate and acetate.CimA encodes a citramalate synthase. Homologs and variants are known andinclude, for example, citramalate synthase (Leptospira biflexa serovarPatoc) gi|116664687|gb|ABK13757.1|(116664687); citramalate synthase(Leptospira biflexa serovar Monteralerio)gi|116664685|gb|ABK13756.1|(116664685); citramalate synthase (Leptospirainterrogans serovar Hebdomadis) gi|116664683|gb|ABK13755.1|(116664683);citramalate synthase (Leptospira interrogans serovar Pomona)gi|116664681|gb|ABK13754.1|(116664681); citramalate synthase (Leptospirainterrogans serovar Australis) gi|116664679|gb|ABK13753.1|(116664679);citramalate synthase (Leptospira interrogans serovar Autumnalis)gi|116664677|gb|ABK13752.1|(116664677); citramalate synthase (Leptospirainterrogans serovar Pyrogenes) gi|116664675|gb|ABK13751.1|(116664675);citramalate synthase (Leptospira interrogans serovar Canicola)gi|116664673|gb|ABK13750.1|(116664673); citramalate synthase (Leptospirainterrogans serovar Lai) gi|116664671|gb|ABK13749.1|(116664671); CimA(Leptospira meyeri serovar Semaranga)gi|119720987|gb|ABL98031.1|(119720987); (R)-citramalate synthasegi|2492795|sp|Q58787.1|CIMA_METJA(2492795); (R)-citramalate synthasegi|22095547|sp|P58966.1|CIMA_METMA(22095547); (R)-citramalate synthasegi|22001554|sp|Q8TJJ1.1|CIMA_METAC(22001554); (R)-citramalate synthasegi|22001553|sp|O26819.1|CIMA_METTH(22001553); (R)-citramalate synthasegi|22001555|sp|Q8TYB1.1|CIMA_METKA(22001555); (R)-citramalate synthase(Methanococcus maripaludis S2)gi|14535858|ref|NP_(—)988138.1|(45358581); (R)-citramalate synthase(Methanococcus maripaludis S2) gi|44921339|emb|CAF30574.1|(44921339);and similar to (R)-citramalate synthase (Candidatus Kueneniastuttgartiensis) gi|91203541|emb|CAJ71194.1|(91203541), each sequenceassociated with the foregoing accession numbers is incorporated hereinby reference.

The proteobacterium Ralstonia eutropha possesses two energy-linked(NiFe) hydrogenases: a membrane hydrogenase and a cytoplasmichydrogenase. The membrane hydrogenase is involved in electrontransport-coupled phosphorylation through coupling to the respiratorychain, whereas the cytoplasmic hydrogenase is able to reduce NAD⁺ togenerate reducing equivalents (Schink et al., Biochim. Biophys. Acta567:315-324, 1979; Schneider et al. Biochim. Biophys. Acta 452:66-80,1976, each of which is incorporated herein by reference in itsentirety). The genes encoding the two hydrogenases are clustered in twoseparate operons together with regulatory genes involved in hydrogenasebiosynthesis on megaplasmid pHG1 (Schultz et al. Science 302:624-627,2003; Schwartz et al. J. Bacteriol. 180:3197-3204, 1998, each of whichis incorporated herein by reference in its entirety). A thirdhydrogenase was identified in R. eutropha and classified as belonging tothe subclass of H₂-sensing (NiFe) hydrogenases (Kleihues et al., J.Bacteriol. 182:2716-2724, 2000, incorporated herein by reference in itsentirety). The third hydrogenase is stable in presence of O₂, CO, andC₂H₂. The rate of hydrogen oxidation of this third hydrogenase is one totwo orders of magnitude lower than that of standard membrane andcytoplasmic hydrogenase. The third hydrogenase contains an active sizesimilar to the initial two hydrogenases. This third hydrogenase isencoded by the hoxB and hoxC genes (large and small subunit,respectively). The hyp genes (hypA1B1F1CDEX) are responsible for thematuration of the third hydrogenase in R. eutropha are located betweenthe membrane hydrogenase genes and hoxA.

Oxygen-tolerant hydrogenases have been identified in Bradyrhizobiumjaponicum (Black et al., 1994), Ra. eutropha (Buhrke et al., 2005; Lenzand Friedrich, 1998), Rhodobacter capsulatus (Elsen et al., 1996;Vignais et al., 2002), Thiocapsa roseopersicina (Kovacs et al., 2005),and Rh. palustris (Rey et al., 2006). Significant heterologous activityof one these hydrogenases has been reported in Synechococcus elongatusPCC7002, with the chromosomal integration of the soluble hydrogenase andaccessory maturation proteins of Ra. eutropha (Xu, 2009).

In a specific embodiment, a microorganism which naturally contains a CO₂fixation enzyme and an ability to use H₂ or formate for reduction isengineered to produce an alcohol. In one embodiment, the alcohol isisobutanol. In another embodiment, the recombinant microorganism isengineered from a Ralstonia sp. to contain a pathway comprising theenzymes and conversion set forth in the following tables. The followingtables set forth reaction pathways for various recombinant microorganismof the disclosure including a list of exemplary genes and homologs andorganism source.

1-Butanol Production Pathway Via Pyruvate

Reaction 1 Pyruvate + Acetyl-CoA −> (R)-citramalate Genes cimA(Methanocaldococcus jannaschii), cimA (Leptospira interrogans) orhomologs thereof Reaction 2 (R)-citramalate −> citraconate Genes leuCD(Leptospira interrogans), leuCD (E. coli) or homologs thereof Reaction 3citraconate −> β-methyl-D-malate Genes leuCD (Leptospira interrogans),leuCD (E. coli) or homologs thereof Reaction 4 β-methyl-D-malate −>2-keto-butyrate Genes leuB (Leptospira interrogans), leuB (E. coli) orhomologs thereof Reaction 5 2-keto-butyrate −> 2-ethylmalate Genes leuA(E. coli) or homologs thereof Reaction 3 2-ethylmalate −>3-ethylmalateGenes leuCD (E. coli) or homologs thereof Reaction 4 3-ethylmalate −>2-ethyl-3-oxosuccinate Genes leuB (E. coli) or homologs thereof Reaction5 2-ethyl-3-oxosuccinate −> 2-keto-valerate Genes (spontaneous) Reaction6 2-keto-valerate −> butrylaldehyde Genes kivd (Lactococcus lactis),kdcA (Lactococcus lactis), PDC1 (Saccharomyces cerevisiae), PDC5(Saccharomyces cerevisiae), PDC6 (Saccharomyces cerevisiae) THI3(Saccharomyces cerevisiae), ARO10 (Saccharomyces cerevisiae)or homologsthereof Reaction 7 butrylaldehyde −> 1-butanol Genes ADH1 (Saccharomycescerevisiae), ADH2 (Saccharomyces cerevisiae), ADH3(Saccharomycescerevisiae), ADH4 (Saccharomyces cerevisiae), ADH5(Saccharomycescerevisiae), ADH6 (Saccharomyces cerevisiae), SFA1 (Saccharomycescerevisiae) or homologs thereof

1-Propanol Production Pathway Via Pyruvate

Reaction 1 Pyruvate + Acetyl-CoA −> (R)-citramalate Genes cimA(Methanocaldococcus jannaschii), cimA (Leptospira interrogans) orhomologs thereof Reaction 2 (R)-citramalate −> citraconate Genes leuCD(Leptospira interrogans), leuCD (E. coli) or homologs thereof Reaction 3citraconate −> β-methyl-D-malate Genes leuCD (Leptospira interrogans),leuCD (E. coli) or homologs thereof Reaction 4 β-methyl-D-malate −>2-keto-butyrate Genes leuB (Leptospira interrogans), leuB (E. coli) orhomologs thereof Reaction 5 2-keto-butyral −> butrylaldehyde Genes kivd(Lactococcus lactis), kdcA (Lactococcus lactis), PDC1 (Saccharomycescerevisiae), PDC5 (Saccharomyces cerevisiae), PDC6 (Saccharomycescerevisiae) THI3 (Saccharomyces cerevisiae), ARO10 (Saccharomycescerevisiae)or homologs thereof Reaction 7 butrylaldehyde −> 1-butanolGenes ADH1 (Saccharomyces cerevisiae), ADH2 (Saccharomyces cerevisiae),ADH3(Saccharomyces cerevisiae), ADH4 (Saccharomyces cerevisiae),ADH5(Saccharomyces cerevisiae), ADH6 (Saccharomyces cerevisiae), SFA1(Saccharomyces cerevisiae) or homologs thereof

3-Methyl-1-Butanol Production Pathway (Via Pyruvate)

Reaction 1 pyruvate −> 2-acetolactate Gene ilvHI (E. coli), ilvNB (E.coli), ilvGM (E. coli), alsS (Bacillus subtilis) or homologs thereofReaction 2 2-acetolactate −> 2,3-dihydroxy-isovalerate Genes ilvC (E.coli) or homologs thereof Reaction 3 2,3-dihydroxy-isovalerate −>2-keto-isovalerate Genes ilvD (E. coli) or homologs thereof Reaction 42-keto-isovalerate −> 2-isopropylmalate Genes leuA (E. coli) or homologsthereof Reaction 5 2-isopropylmalate −> 3-isopropylmalate Genes leuCD(E. coli) or homologs thereof Reaction 6 3-isopropylmalate −>2-isopropyl-3-oxosuccinate Genes leuB (E. coli) or homologs thereofReaction 7 2-isopropyl-3-oxosuccinate −> 2-ketoisocaproate Gene(spontaneous) Reaction 8 2-ketoisocaproate −> 3-methylbutyraldehydeGenes kivd (Lactococcus lactis), kdcA (Lactococcus lactis), PDC1(Saccharomyces cerevisiae), PDC5 (Saccharomyces cerevisiae), PDC6(Saccharomyces cerevisiae) THI3 (Saccharomyces cerevisiae), ARO10(Saccharomyces cerevisiae)or homologs thereof Reaction 93-methylbutyraldehyde −> 3-methyl-1-butanol Genes ADH1 (Saccharomycescerevisiae), ADH2 (Saccharomyces cerevisiae), ADH3(Saccharomycescerevisiae), ADH4 (Saccharomyces cerevisiae), ADH5(Saccharomycescerevisiae), ADH6 (Saccharomyces cerevisiae), SFA1 (Saccharomycescerevisiae) or homologs thereof

Isobutanol Production Pathway (Via Pyruvate)

Reaction 1 pyruvate −> 2-acetolactate Genes ilvHI (E. coli), ilvNB (E.coli), ilvGM (E. coli), alsS (Bacillus subtilis) or homologs thereofReaction 2 2-acetolactate −> 2,3-dihydroxy-isovalerate Genes ilvC (E.coli) or homologs thereof Reaction 3 2,3-dihydroxy-isovalerate −>2-keto-isovalerate Genes ilvD (E. coli) or homologs thereof Reaction 42-keto-isovalerate −> isobutrylaldehyde Genes kivd (Lactococcus lactis),kdcA (Lactococcus lactis), PDC1 (Saccharomyces cerevisiae), PDC5(Saccharomyces cerevisiae), PDC6 (Saccharomyces cerevisiae) THI3(Saccharomyces cerevisiae), ARO10 (Saccharomyces cerevisiae) or homologsthereof Reaction 5 isobutrylaldehyde −> isobutanol Genes ADH1(Saccharomyces cerevisiae), ADH2 (Saccharomyces cerevisiae),ADH3(Saccharomyces cerevisiae), ADH4 (Saccharomyces cerevisiae),ADH5(Saccharomyces cerevisiae), ADH6 (Saccharomyces cerevisiae), SFA1(Saccharomyces cerevisiae) or homologs thereof

As previously discussed, general texts which describe molecularbiological techniques useful herein, including the use of vectors,promoters and many other relevant topics, include Berger and Kimmel,Guide to Molecular Cloning Techniques, Methods in Enzymology Volume 152,(Academic Press, Inc., San Diego, Calif.) (“Berger”); Sambrook et al.,Molecular Cloning—A Laboratory Manual, 2d ed., Vol. 1-3, Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y., 1989 (“Sambrook”) andCurrent Protocols in Molecular Biology, F. M. Ausubel et al., eds.,Current Protocols, a joint venture between Greene Publishing Associates,Inc. and John Wiley & Sons, Inc., (supplemented through 1999)(“Ausubel”). Examples of protocols sufficient to direct persons of skillthrough in vitro amplification methods, including the polymerase chainreaction (PCR), the ligase chain reaction (LCR), Q□-replicaseamplification and other RNA polymerase mediated techniques (e.g.,NASBA), e.g., for the production of the homologous nucleic acids of thedisclosure are found in Berger, Sambrook, and Ausubel, as well as inMullis et al. (1987) U.S. Pat. No. 4,683,202; Innis et al., eds. (1990)PCR Protocols: A Guide to Methods and Applications (Academic Press Inc.San Diego, Calif.) (“Innis”); Arnheim & Levinson (Oct. 1, 1990) C&EN36-47; The Journal Of NIH Research (1991) 3: 81-94; Kwoh et al. (1989)Proc. Natl. Acad. Sci. USA 86: 1173; Guatelli et al. (1990) Proc. Nat'l.Acad. Sci. USA 87: 1874; Lomell et al. (1989) J. Clin. Chem. 35: 1826;Landegren et al. (1988) Science 241: 1077-1080; Van Brunt (1990)Biotechnology 8: 291-294; Wu and Wallace (1989) Gene 4:560; Barringer etal. (1990) Gene 89:117; and Sooknanan and Malek (1995) Biotechnology 13:563-564. Improved methods for cloning in vitro amplified nucleic acidsare described in Wallace et al., U.S. Pat. No. 5,426,039. Improvedmethods for amplifying large nucleic acids by PCR are summarized inCheng et al. (1994) Nature 369: 684-685 and the references citedtherein, in which PCR amplicons of up to 40 kb are generated. One ofskill will appreciate that essentially any RNA can be converted into adouble stranded DNA suitable for restriction digestion, PCR expansionand sequencing using reverse transcriptase and a polymerase. See, e.g.,Ausubel, Sambrook and Berger, all supra.

EXAMPLES

DNA polymerase KOD for PCR reactions can be purchased from EMD Chemicals(San Diego, Calif.). All restriction enzymes and Antarctic phosphatasecan be obtain from New England Biolabs (Ipswich, Mass.). Rapid DNAligation kit is available from Roche (Manheim, Germany).Oligonucleotides can be ordered from Operon (Huntsville, Ala.). Allantibiotics and reagents in media are available from either SigmaAldrich (St. Louis, Mo.) or Fisher Scientifics (Houston, Tex.).

Bacterial Strains.

Escherichia coli BW25113 (rrnB_(T14) ΔlacZ_(WJ16) hsdR514 ΔaraBAD_(AH33)ΔrhaBAD_(LD78)) was designated as the wild-type (WT) (Datsenko andWanner, Proc. Natl. Acad. Sci. USA 97, 6640-6645, 2000) for comparison.In some experiments for isobutanol, JCL16 (rrnB_(T14) ΔlacZ_(WJ16)hsdR514ΔaraBAD_(AH33) ΔrhaBAD_(LD78)/F′ (traD36, proAB+, lacIqZΔ11/115)) was used as wild-type (WT). Host gene deletions of metA, tdh,ilvB, ilvI, adhE, pta, ldhA, and pflP were achieved with P1 transductionusing the Keio collection strains (Baba et al., Mol. Systems Biol. 2,2006) as donor. The kan^(R) inserted into the target gene region wasremoved with pCP20 (Datsenko and Wanner, supra) in between eachconsecutive knock out. Then, removal of the gene segment was verified bycolony PCR using the appropriate primers. XL-1 Blue (Stratagene, LaJolla, Calif.) was used to propagate all plasmids.

Plasmid Construction.

pSA40, pSA55, and pSA62 were designed and constructed as describedelsewhere herein. The lacI gene was amplified with primers lad SacI fand lacI SacI r from E. coli MG 1655 genomic DNA. The PCR product wasthen digested with SacI and ligated into the pSA55 open vector cut withthe same enzyme behind the promoter of the ampicillin resistance gene,creating pSA55I.

The gene tdcB was amplified with PCR using primers tdcB f Acc65 and tdcBr SalI from the genomic DNA of E. coli BW25113 WT. The resulting PCRproduct was gel purified and digested with Acc65 and SalI. The digestedfragment was then ligated into the pSA40 open vector cut with the samepair of enzymes, creating pCS14.

To replace the replication origin of pCS14 from colE1 to p15A, pZA31-lucwas digested with SacI and AvrII. The shorter fragment was gel purifiedand cloned into plasmid pCS14 cut with the same enzymes, creating pCS16.

The operon leuABCD was amplified using primers A106 and A109 and E. coliBW25113 genomic DNA as the template. The PCR product was cut with SalIand BglII and ligated into pCS16 digested with SalI and BamHI, creatingpCS20.

To create an expression plasmid identical to pSA40 but with p15A origin,the p15A fragment obtained from digesting pZA31-luc with SacI and AvrIIwas cloned into pSA40 open vector cut with the same restriction enzymes,creating pCS27.

The leuA* G462D mutant was constructed using SOE (Splice Overlapextension) with primers G462Df and G462Dr and the E. coli BW25113 WTgenomic DNA as a template to obtain leuA*BCD. Then the SOE product wasdigested and cloned into the restriction sites Acc65 and XbaI to createPZE_leuABCD. The resulting plasmid was next used as a template to PCRout the leuA*BCD using primers A106 and A109. The product was cut withSalI and BglII and ligated into pCS27 digested with SalI and BamHI,creating pCS48.

The gene ilvA was amplified from E. coli BW25113 WT genomic DNA withprimers A110 and A112. Next, it was cut with Acc65 and XhoI and ligatedinto the pCS48 open vector digested with Acc65 and SalI, creating pCS51.

The gene tdcB from the genomic DNA of E. coli BW25113 WT was amplifiedwith PCR using primers tdcB f Acc65 and tdcB r SalI. The resulting PCRproduct was gel purified, digested with Acc65 and SalI and then ligatedinto the pCS48 open vector cut with the same pair of enzymes, creatingpCS50.

WT thrABC was amplified by PCR using primers thrA f Acc65 and thrC rHindIII. The resulting product was digested with Acc65 and HindIII andcloned into pSA40 cut with the same pair of enzymes, creating pCS41.

To replace the replication origin of pCS41 from colE1 to pSC101,pZS24-MCS1 was digested with SacI and AvrII. The shorter fragment wasgel purified and cloned into plasmid pCS41 cut with the same enzymes,creating pCS59.

The feedback resistant mutant thrA* was amplified by PCR along with thrBand thrC from the genomic DNA isolated from the threonine over-producerATCC 21277 using primers thrA f Acc65 and thrC r HindIII. The resultingproduct was digested with Acc65 and HindIII and cloned into pSA40 cutwith the same pair of enzymes, creating pCS43.

To replace the replication origin of pCS43 from colE1 to pSC101,pZS24-MCS1 was digested with SacI and AvrII. The shorter fragment wasgel purified and cloned into plasmid pCS43 cut with the same enzymes,creating pCS49.

Branched-chain amino-acid aminotransferase (encoded by ilvE) andtyrosine aminotransferase (encoded by tyrB) were deleted by P1transduction from strains disclosed in Baba et al.

To clone the L-valine biosynthesis genes i) ilvIHCD (EC) and ii) als(BS) along with ilvCD (EC), the low copy origin of replication (ori)from pZS24-MCS1 was removed by digestion with SacI and AvrII, andligated into the corresponding sites of i) pSA54 and ii) pSA69 to createplasmid pIAA1 and pIAA11, respectively.

To clone kivd from L. lactis and ADH2 from S. cerevisiae, the ColE1 onof pSA55 was removed by digestion with SacI and AvrII and replaced withthe p15A on of pSA54 digested with the same restriction enzymes tocreate pIAA13. To better control the expression of these genes, lad wasamplified from E. coli MG 1655 genomic DNA with KOD polymerase usingprimers lacISaclf and lacISacIr and ligated into the SacI site of pCS22to be expressed along with the ampicillin resistance gene, bla, andcreate plasmid pIAA12.

In order to overexpress the leuABCD operon in BW25113/F′ from thechromosome, the native promoter and leader sequence was replaced withthe P_(LlacO-1) promoter. The P_(LlacO-1) promoter was amplified frompZE12-luc with KOD polymerase using primers lacO1KanSOEf andlacO1LeuAlr. The gene encoding resistance to kanamycin, aph, wasamplified from pKD13 using primers KanLeuO1f and KanlacO1SOEr. 1 μL ofproduct from each reaction was added as template along with primersKanLeuO2f and lacO1LeuA2r, and was amplified with KOD polymerase usingSOE. The new construct was amplified from the genomic DNA of kanamycinresistant clones using primers leuKOv1 and leuKOv2 and sent out forsequence verification to confirm the accuracy of cloning. To overexpressthe leuABCD operon from plasmid, the p15A on from pSA54 was removed withSacI and AvrII and ligated into the corresponding sites of pCS22 (ColE1,Cm^(R), P_(LlacO-1):leuABCD) to create plasmid pIAA2. In order fortighter expression, lad was amplified and ligated as describedpreviously for pIAA12 into pCS22 to be expressed along with thechloroamphenicol resistance gene, cat, and create plasmid pIAA15.Plasmid pIAA16 containing leuA(G1385A) encoding for IPMS (G462D) wascreated by ligating the 5.5 kb fragment of pIAA15 digested with XhoI andNdeI and ligating it with the 2.3 kb fragment of pZE12-leuABCD (ColE1,Amp^(R), P_(LlacO-1): leuA(G1385A)BCD) cut with the same restrictionenzymes. To control for expression level, the RBS was replaced in pIAA15to match that of pIAA16. To do this, the 5.6 kb fragment of pIAA16 fromdigestion with HindIII and NdeI was ligated with the 2.2 kb fragment ofpIAA15 digested with the same enzymes to create pIAA17.

Media and Cultivation.

Certain strains were grown in a modified M9 medium (6 g Na₂HPO4, 3 gKH₂PO₄, 1 g NH₄Cl, 0.5 g NaCl, 1 mM MgSO₄, 1 mM CaCl₂, 10 mg Vitamin B1per liter of water) containing 10 g/L of glucose, 5 g/L of yeastextract, and 1000× Trace Metals Mix A5 (2.86 g H₃BO₃, 1.81 g MnCl₂.4H₂O,0.222 g ZnSO₄.7H₂O, 0.39 g Na₂MoO₄.2H₂O, 0.079 g CuSO₄.5H₂O, 49.4 mgCo(NO₃)₂.6H₂O per liter water) inoculated 1% from 3 mL overnightcultures in LB into 10 mL of fresh media in 125 mL screw cap flasks andgrown at 37° C. in a rotary shaker for 4 hours. The culture was theninduced with 1 mM IPTG and grown at 30° C. for 18 hours. Antibioticswere added as needed (ampicillin 100 μg/mL, chloroamphenicol 35 μg/mL,kanamycin 50 μg/mL).

For some alcohol fermentation experiments, single colonies were pickedfrom LB plates and inoculated into 3 ml of LB media with the appropriateantibiotics (ampicillin 100 μg/ml, kanamycin 50 μg/ml, and spectinomycin50 μg/ml). The overnight culture grown in LB at 37° C. in a rotaryshaker (250 rpm) was then inoculated (1% vol/vol) into 20 ml of M9medium (6 g Na₂HPO₄, 3 g KH₂PO₄, 0.5 g NaCl, 1 g NH₄Cl, 1 mM MgSO₄, 10mg vitamin B1 and 0.1 mM CaCl₂ per liter of water) containing 30 g/Lglucose, 5 g/L yeast extract, appropriate antibiotics, and 1000× TraceMetal Mix A5 (2.86 g H₃BO₃, 1.81 g MnCl₂.4H₂O, 0.222 g ZnSO₄.7H₂O, 0.39g Na₂MoO₄.2H₂O, 0.079 g CuSO₄.5H₂O, 49.4 mg Co(NO₃)₂.6H₂O per literwater) in 250 ml conical flask. The culture was allowed to grow at 37°C. in a rotary shaker (250 rpm) to an OD₆₀₀ of 0.4˜0.6, then 12 ml ofthe culture was transferred to a 250 ml screw capped conical flask andinduced with 1 mM IPTG. The induced cultures were grown at 30° C. in arotary shaker (240 rpm). Samples were taken throughout the next three tofour days by opening the screwed caps of the flasks, and culture brothswere either centrifuged or filtered to retrieve the supernatant. In someexperiments as indicated, 8 g/L of threonine was added directly into thecell culture at the same time of induction.

α-keto acid experiments were done under oxygen ‘rich’ conditions unlessotherwise noted. For oxygen rich experiments, 10 mL cultures in 250 mLbaffled shake flasks were inoculated 1% from 3 mL overnight cultures inLB. For oxygen poor experiments, 10 mL cultures were inoculated in 125mL screw caps. All cultures were grown at 37° C. for 4 hours and inducedwith 1 mM IPTG and harvested after 18 hrs of growth at 30° C.

Metabolite Detections.

The produced alcohol compounds can be quantified by a gas chromatograph(GC) equipped with flame ionization detector. The system includes model5890A GC (Hewlett-Packard, Avondale, Pa.) and a model 7673A automaticinjector, sampler and controller (Hewlett-Packard). Supernatant ofculture broth (0.1 ml) is injected in split injection mode (1:15 splitratio) using methanol as the internal standard.

The separation of alcohol compounds is carried out by A DB-WAX capillarycolumn (30 m, 0.32 mm-i.d., 0.50 μm-film thickness) purchased fromAgilent Technologies (Santa Clara, Calif.). GC oven temperature isinitially held at 40° C. for 5 min and raised with a gradient of 15°C./min until 120° C. It is then raised with a gradient of 50° C./minuntil 230° C. and held for 4 min. Helium is used as the carrier gas with9.3 psi inlet pressure. The injector and detector are maintained at 225°C. 0.5 ul supernatant of culture broth is injected in split injectionmode with a 1:15 split ratio. Methanol is used as the internal standard.

For other secreted metabolites, filtered supernatant is applied (20 ul)to an Agilent 1100 HPLC equipped with an auto-sampler (AgilentTechnologies) and a BioRad (Biorad Laboratories, Hercules, Calif.)Aminex HPX87 column (5 mM H₂SO₄, 0.6 ml/min, column temperature at 65°C.). Glucose is detected with a refractive index detector, while organicacids are detected using a photodiode array detector at 210 nm.Concentrations are determined by extrapolation from standard curves.

For other secreted metabolites, filtered supernatant is applied (0.02ml) to an Agilent 1100 HPLC equipped with an auto-sampler (AgilentTechnologies) and a BioRad (Biorad Laboratories, Hercules, Calif.)Aminex HPX87 column (0.5 mM H₂SO₄, 0.6 mL/min, column temperature at 65°C.). Glucose is detected with a refractive index detector while organicacids are detected using a photodiode array detector at 210 nm.Concentrations are determined by extrapolation from standard curves.

Cyanobacteria encompass a large group of photosynthetic microorganismsthat vary widely in morphology, habitat, and physiology. Included inthis group is the unicellular Synechococcus sp. strain PCC 7942(previously Anacystis nidulans R2), which is one of the fewcyanobacterial strains which have been well-characterized in terms ofphysiology, biochemistry, and genetics. As stated previously, S.elongatus PCC7942 has been engineered to produce up to 1.1 g/L ofisobutryaldehyde from CO₂ (see, e.g., Atsumi et al., 2009) by utilizingthe microorganism's photosynthesis and CBB cycle. In addition to S.elongatus PCC7942, other cyanobacterial strains can be used. Forexample, S. elongatus PCC7002 has the ability to grow heterotrophicallyon glycerol and has a shorter generation time of 4 hr compared to 6.4 hrfor S. elongatus PCC7942.

In order to engineer S. elongatus to utilize H₂ as an electron donor,strains that express hydrogenase genes from Ra. eutropha, B. japonicum,R. capsulatus, and Rh. palustris are constructed by chromosomalinsertion of the expression cassettes into neutral site 1 (NSI). Anexpression cassette is thus created by cloning the individual genes intothe NSI-targeting vector, pAM2991 under the IPTG-inducible Ptrcpromoter. Methods for measuring in vitro and in vivo hydrogenaseactivity have been well-established (Vignais and Billoud, 2007) and canbe used to determine the best hydrogenase for a particular system.

To improve the H₂ uptake rate of the hydrogenases error prone PCR can beused on one of the oxygen-tolerant hydrogenases (e.g., from Ra.eutropha). Under conditions where the photosynthetic activity ofSynechococcus is relatively low (i.e., low light conditions), thefastest growing transformants can be analyzed for improvements in H₂uptake (Vignais and Billoud, 2007). Other approaches can be used tocapitalize on the loss of autotrophic growth, but maintenance ofheterotrophic growth of a Ra. eutropha ΔhoxFUYG hydrogenase mutant(Massanz, 1998). An expression library of mutant, oxygen-toleranthydrogenases created by error-prone PCR from Ra. eutropha and/or otherspecies will be transformed into the Ra. eutropha ΔhoxFUYG hydrogenasemutant. Grown under lithoautotrophic conditions, the fastest growingtransformants express mutant hydrogenases with improved H₂ uptake and/oractivity, which can be ascertained by H₂ uptake assays (Vignais andBilloud, 2007). The genes that express these mutant hydrogenases withimproved H₂ uptake activity can be cloned into the NSI-targeting vectorand introduced into S. elongatus for expression.

In order to engineer S. elongatus to oxidize formate for the productionof reducing equivalents, formate dehydrogenases (FDHs) areheterologously expressed in this microorganism. FDHs have been proven tobe the most promising candidate for the development of NAD+ regenerationsystems in organic synthesis for production of high-added-value productslargely due to their wide pH-optimum (pH 6.0-9.0) and to thenon-reversibility of enzymes (Burton, 2003; Hummel and Kula, 1989;Shaked et al., 1980; Wichmann and Vasic-Racki, 2005). Of the FDHs thathave been studied, the one from Candida boidinii is the most commonlyused for the development of NAD+ regeneration systems (Ohshima et al.,1985). Studies on C. boidinii FDH have identified mutations that conferaltered cofactor specificity (Rozzell, 2004), improved catalyticactivity (Slusarczyk, 2003), and enhanced chemical stability(Slusarczyk, 2003; Felber, 2001). Using various optimized FDH, theactivity in S. elongates can be optimized, especially in altering thecofactor specificity from NAD(H) to NADP(H) because S. elongatus has apreference for NADP(H) (Tamoi et al., 2005).

Several FDHs have been integrated into the NSI site of S. elongatusPCC7942. The genes that encode the wild type and D195S/Y196H doublemutant FDH from C. boidinii and the FDH from M. thermoacetica were eachcloned into the NSI-targeting vector, under the IPTG-inducible Ptrcpromoter: The D195S/Y196H double mutation was utilized because itresults in a FDH with altered cofactor specificity from NAD(H) toNADP(H). The FDH gene from Moorella thermoacetica, encoded byMoth_(—)2314, has been indicated to encode for an enzyme withformate:NADP+ oxidoreductase activity. This enzyme was chosen because ofits cofactor preference.

In addition to the FDHs, other genes were also heterologously expressedto optimize formate utilization. To ensure efficient formate uptake, aformate transporter encoded by focA from E. coli was also overexpressed.Furthermore, to specifically generate NADPH from formate oxidization,several transhydrogenases including pntAB and udhA from E. coli havebeen introduced in combination with wild type NAD+-dependent C. boidiniiFDH. By using enzymatic assays of crude cyanobacterial cell lysates, aswell as HPLC measurements of formate consumption in flask culture, theco-expression of E. coli focA, C. boidinii wild type FDH, and E. colipntAB enable S. elongatus to consume formate at a significant rate.

To improve CO₂ fixation, an additional copy of the CBB cycle genes,rbcLS, were integrated into the chromosome of the isobutyraldehyde S.elongatus PCC7942 production strain, resulting in a 2-fold increase inisobutyraldehyde (Atsumi et al., 2009). This example, along withsuccessful examples of fructose-1,6/sedoheptulose-1,7-bisphosphataseoverexpression (Miyagawa et al. 2001; Ma et al. 2005), illustrate thatoverexpression of CBB enzymes can enhance photosynthesis efficiency,growth characteristics, and biofuel production. Additional copies ofmany of the CBB cycle genes have been integrated into the NSI and NSIIsites of S. elongatus PCC7942. Genes that have been integrated includethose that encode for fructose-1-6-bisphosphatase 1(Synpcc7942_(—)2335), ribulose-phosphate 3-epimerase(Synpcc7942_(—)0604), sedoheptulose bisphosphatase (Synpcc7942_(—)0505),ribose 5-phosphate isomerase (Synpcc7942_(—)0584), phosphoribulokinase(Synpcc7942_(—)0977), and the E. coli transketolase, tktA.

In cyanobacteria and higher plants, CO₂ fixation is regulated by variousregulation pathways, which can be divided into two major categories:transcriptional and posttranslational. In both cases, the redox statusof the photosynthetic electron transportation chain has been proposed toplay an important role in light sensing as the signaling input pathway(Buchanan and Balmer, 2005; Golden, 1995). Once received, the lightsignal is then relayed from the photosynthetic machinery to othercellular mediators, including various proteins in theferredoxin/thioredoxin system and KaiABC oscillator system (Buchanan andBalmer, 2005; Ivleva et al., 2006; Lindahl and Florencio, 2003; Schmitzet al., 2000).

Transcription of most of the CBB cycle genes are significantlysuppressed in the dark cycle (Ito et al., 2009; Nakahira et al., 2004).One of the most extensively studied regulation systems in S. elongatusPCC7942 is the KaiABC circadian rhythm oscillator system, which governsthe global transcription profile in a diurnal cyclic fashion (Ishiura etal., 1998; Johnson et al., 2008). Recent studies have shown thattranscriptional activity from most of the promoters in S. elongatusdisplayed substantial fluctuation over a day/night cycle (Ito et al.,2009; Liu et al., 1995; Smith and Williams, 2006). Moreover, the overallorganization of the S. elongatus chromosome undergoes cyclic change(Nakahira et al., 2004; Smith and Williams, 2006), which may affect theexpression level of both endogenous and genome-integrated heterogeneousproduction pathways. Previous studies have shown that disruption of thekaiABC gene cluster delivered the arrhythmia phenotype in S. elongatusPCC7942, although the average expression level of each individual genein the genome was not dramatically altered (Ito et al., 2009). This andsimilar arrhythmic strains may be favored for CO, fixation in the dark,due to their steady global gene expression levels regardless of changinglight condition. In addition, to maintain CBB gene expression at a highlevel, enzymes such as RuBisCO, phosphoribulokinase (PRK), andglyceraldehyde-3-phosphate dehydrogenase (GAPDH) can be constitutivelyoverexpressed.

Posttranslational level (or protein level) regulation represents anotherlayer of light/dark regulation of CO₂ fixation on top of transcriptionalregulation. The exchange of dithiol/disulfide status controlled by theferredoxin/thioredoxin system is one of these conservedposttranslational regulation mechanism utilized by chloroplasts ofplants, algae, as well as photosynthetic microorganisms, to adjustenzyme activities according to light condition (Buchanan et al., 1980;Pfannschmidt et al., 2000; Buchanan et al., 2002; Lindahl et al., 2003).In light conditions, ferredoxin receives electrons from Photosystem I(PS I) and transfers them to thioredoxin (Trx), mediated by the enzymeferredoxin-Trx reductase (FTR). Thioredoxin can then reduce disulfidebonds formed between cysteine residues within target enzymes and thusmodulate their activities.

In contrast to higher plants, most enzymes in the CBB cycle ofcyanobacterium Synechocystis sp. PCC 6803 are not directly regulated bythe ferredoxin/thioredoxin system (Lindahl and Florencio, 2003).Specifically, although fructose-1,6-bisphosphatase (FBPase),NADP+-glycerolaldehyde-3-phosphate dehydrogenase (NADP+-GAPDH), andphosphoribulokinase (PRK) are greatly suppressed in the dark conditionby redox regulation in higher plants (Buchanan, 1980), similar redoxregulation of these three enzymes have been suggested to be absent incyanobacteria Synechocystis sp. PCC 6803 and Synechococcus elongatusPCC7942 by biochemical studies (Tamoi et al., 1996; Tamoi et al., 1998).Consistently, it has also been indicated from amino acid sequencealignment that the potential regulatory cysteine residues are missing incyanobacterial NADP+-GAPDH and FBPase (Tamoi et al., 1996; Tamoi et al.,1998).

Thus, removing ferredoxin/thioredoxin-mediated redox regulation of theCBB enzymes in cyanobacteria can be performed. RuBisCO has beensuggested to be a conserved ferredoxin/thioredoxin target (Lindahl andFlorencio, 2003). Fortunately, with a C172A mutation in the RuBisCO ofSynechocystis sp. strain PCC6803, the inhibitory effect of oxidants thatreact with the vicinal thiols in RuBisCO is alleviated (Marcus et al.,2003). Since the regulatory cysteines are conserved among cyanobacteriaspecies, these observations provided useful information for proteinengineering in the construction of a redox-resistant RuBisCO in S.elongatus PCC7942.

Besides the universal redox regulation system shared by allphotosynthetic organisms, cyanobacterial cells also possess other uniqueposttranslational mechanisms to regulate CO₂ fixation. For example,protein CP12 in S. elongatus PCC7942 has been found to form a complexwith RuBisCO and GAPDH to inhibit their activities in the dark (Wedeland Soli, 1998). Furthermore, the formation of this complex isdynamically regulated by CP12, which is able to sense the NAD(H)/NADP(H)ratio under light/dark conditions (Tamoi et al., 2005). Incyanobacteria, mutations that prevent CP12 expression had no effectduring conditions of continuous light, but resulted in inhibited growthin light/dark diurnal conditions presumably due to a carbon metabolismdisorder related to leaky CBB cycle activity in the dark (Tamoi et al.,2005). By inactivating CP12 using genetic or protein engineeringapproaches, formation of the inhibitory complex could be eliminated,releasing the CBB cycle from light/dark regulation.

As a chemolithoautotroph, Ra. eutropha is able to derive its energy andreducing power from inorganic compounds or elements, such as H₂ orformate, to drive CO₂ fixation through the CBB cycle.

Ra. eutropha employs native hydrogen utilization pathways when itundergoes chemoautotrophic growth. Two types of hydrogen utilizationpathways run in parallel to fuel the CO₂-fixing CBB cycle with ATP andNADPH: A membrane-bound hydrogenase (MBH), which oxidizes H2 and feedselectrons into the respiratory chain to generate ATP; and also a solublehydrogenase (SH), which directly uses NAD(P)+ as an electron acceptor toproduce NAD(P)H at the expense of H₂. In addition, severaltranshydrogenases convert NADH into NADPH in order to meet the NADPHneeds required by the CBB cycle (Cramm, 2009; Pohlmann et al., 2006).Ra. eutropha hydrogenases belong to a family of (NiFe) bidirectionalhydrogenases. However, unlike most of the members in the family, whichare sensitive to very low oxygen concentrations, Ra. eutrophahydrogenases are relatively oxygen tolerant, consistent with the aerobicphysiological nature of this organism.

Similarly, formate can serve as both an electron donor and carbon sourceto sustain autotrophic growth of Ra. eutropha. A membrane-bound formatedehydrogenase oxidizes formate and transports the electrons intorespiratory chain; and a soluble formate dehydrogenase uses NAD+ as theelectron acceptor. The CO₂ produced from formate oxidization is thenassimilated (Cramm, 2009; Pohlmann et al., 2006).

CO₂ is fixed through the CBB cycle in Ra. eutropha to pyruvate. Byengineering alsS from B. subtilis, ilvCD and yqhD from E. coli, and kivdfrom L. lactis into Ra. eutropha autotrophic isobutanol synthesis can beobtained.

To enhance isobutanol production efficiency, competing pathways thatdissipate reducing equivalence or drain carbon flux can be eliminated.In Ra. eutropha, a prominent example would be the PHA productionpathway. The cells can naturally accumulate up to about 70% PHA (of thecell mass), even in autotrophic conditions with CO₂ and H₂ as substrates(Tanaka et al., 1995), which utilizes a large portion of carbon sourceand NADPH pools. Fortunately, the PHA production pathway is very wellknown and genetic manipulation tools to perform knock-out studies areavailable.

To achieve high titer levels of isobutanol production, it is beneficialto isolate a mutant that has a higher tolerance to isobutanol. Thegram-negative Ra. eutropha appears to have comparable solvent toleranceto that of E. coli. Given the success in developing and characterizingE. coli strains that can tolerate up to 8 g/L isobutanol, similarmutagenesis approaches can be utilized in addition to solventchallenging selection. Furthermore, based on high-throughput genomic DNAsequencing of the solvent tolerant strains generated by our group aswell as others, rational strain engineering approaches may also becomeavailable.

Purple bacteria, such as Rhodopsudomonas and Rhodobacter, demonstratelithoautotrophic and chemoautotrophic growth with many organic andinorganic electron donors, including hydrogen and formate. Thesemicroorganisms are able to grow in a mineral medium in the dark at theexpense of hydrogen, oxygen, and CO₂. Although their growth is sensitiveto O₂, the presence of methanol in the medium can improve oxygentolerance (Siefert and Pfennig, 1979). Given these factorablecharacteristics Rh. palustris can be a host for isobutanol synthesisfrom CO₂ and H₂ or formate.

Either co-replicated plasmids or chromosome integration is used toexpress enzymes of the isobutanol pathway. Specifically, alsS from B.subtilis, ilvCD and yqhD from E. coli, and kivd and yqhD from L. lactiscan be engineered into the microorganism. Functional expression of thepathway can be examined by enzyme assays and by measuring the productionof isobutanol under chemoheterotrophic growth conditions. Isobutanolproduction in Rh. palustris can be investigated in electron-autotrophicconditions with hydrogen or formate as the electron donor.Electron-autotrophic biofuel production is performed in the dark undereither aerobic or microaerobic conditions.

Rh. palustris is able to sense redox status and ATP levels, and is thusable to change metabolic modes according to changes in cultureconditions (Larimer et al., 2004). Experimental evidence has shown thatsingle-gene deletions of cbbRRS results in a significant reduction intotal RuBisCO activity, which indicates that the cbbRRS is essential forRuBisCO expression (Romagnoli and Tabita, 2006). Therefore, in order toimprove or maintain CBB cycle activity during different metabolicconditions, upregulation of cbbRRS by overexpression or modify the PASdomains of cbbR can be performed to make it more efficient in catalyzingthe phosphorylation cascade.

To select host organisms for further development the host strain will beexposed to mutagens, and then the surviving culture will be enriched forchemoautotrophic growth. Through several generation of metabolicevolution, the fast-growing mutants will dominate the culture. Sincefast growth indicates high carbon fixation rates, these mutants mostlikely will demonstrate improved CBB pathway efficiency and will besubject to further engineering, such as deregulation and overexpressionof CBB pathway enzymes.

In addition, the metabolite profile of electron-autotrophic productionconditions is analyzed with HPLC-DAD and GC-FID. Once the majorby-products are confirmed, the critical genes that are responsible fortheir formation are identified for inactivation. The isobutanolproduction efficiency is also controlled by the reducing power supply.Overexpression of NAD(P)H-generating hydrogenases and formatehydrogenases can improve energy input and biofuel production efficiencyin the system.

H₂ can be produced by the electrolysis of water. In conventionalelectrolyzers, 25˜30% potassium hydroxide is added to facilitate thedissociation of water into H⁺ and OH⁻. It is however corrosive tooperate electrolysis in a basic environment. As a result, solid polymerelectrolyte membranes (SPE) or proton exchange membranes (PEM) weredeveloped to aid in the splitting of water in a neutral environment. TheSPE or PEM electrolyzer, as the name implies, contains a polymer as amembrane separating the cathode side from the anode side. The formationof O₂ and H₂ is separated into two compartments by a solid electrolytemembrane. One of the most commonly used solid electrolytes is nafion.The solvated SO³⁻ ions act as the proton carriers, which carries protonsfrom the anode to the cathode, which is later reduced to H₂. Theefficiency of the SPE membrane electrolyzer is estimated to be about80˜94%.

The electro-autotrophic fermentation system uses gas-phase substrates tosupply for carbon and reducing power needs. When the gases are fed intothe bioreactor, the solubility of the gases will normally be very low.Fortunately, the electro-autotrophic organisms of the disclosure havelower metabolic activities compared to conventional sugar-basedfermentations. In order to minimize energy consumption, impellers areavoided which are energy intensive. Instead, mass transfer and cellsuspension will be used to optimize the gas circulation rate. The gasstream is replenished and recycled to complete a closed system with noH₂ outlet. In addition, the ratio of the three components (H₂, O₂, andCO₂) is optimized for growth and productivity. Optimization of pH,temperature, medium components (among others) is also performed and iswithin the skill in the art.

For isobutanol purification, several conventional n-butanol separationtechnologies are known (e.g. gas-stripping and adsorption).

To develop Ralstonia eutropha as an isobutanol producer the valinebiosynthetic pathway was strengthened to make enough2-KIV(2-ketoisovalerate), which is the precursor for isobutanol. Thesynthetic pathway genes to convert 2-KIV into isobutanol were thenengineered into the microorganism.

Since isobutanol is produced by decarboxylation and subsequent reductionof 2-Ketoisovalerate (2-KIV), an intermediate in valine biosynthesis, itis essential to enhance metabolic flux through valine biosynthesispathway in the host. Two different approaches as shown in FIG. 1F wereundertaken. As shown in FIG. 1F one approach taked was to strengthennatural valine biosynthetic pathway in Ralstonia, while a secondapproach taken was to introduce heterologous genes for valinebiosynthesis pathway. In the genome of Ralstonia eutropha, the naturallyexisting 2-KIV biosynthesis pathway genes include ilvBHC and ilvD genesat separate loci. These natural genes were overexpressed withinRalstonia eutropha by chromosomal knocking-in of a strong phaC promoterin front of the corresponding operons as shown in FIG. 1F. Anotherapproach introduced foreign genes for valine biosynthesis pathway. Inthe second method the artificial operon of alsS from B. subtilis andilvCD from Escherichia coli was used under the phaC promoter ofRalstonia eutropha. This artificial operon was introduced intochromosomal phaB2-phaC2 loci by conjugational double-crossoverintegration as shown in FIG. 1F.

To verify the enhanced activities of 2-KIV production enzymes, theenzyme activities of these 3 enzymes was analyzed. As shown in FIG. 2C,compared to wild type Ralstonia eutropha strain H16, cells(LH66) withmodifications in natural valine biosynthesis genes using the phaCpromoter showed around 9 fold, 3 fold, and 4 fold increase of ilvBH,ilvC, ilvD activities, respectively. The alsS gene from Bacillussubtilis have higher catalytic activity and affinity to pyruvate andwere expected to be more productive. As expected the strain (LH67),which has an integrated artificial operon of alsS from B. subtilis andilvCD from Escherichia coli driven by phaC promoter in the genome,showed much better enzyme activities in all three enzymes. Therefore,this LH67 strain was used for the construction of isobutanol productionstrain in Ralstonia eutropha.

For the efficient conversion of 2-KIV into isobutanol, two moreenzymatic reactions catalyzed by a 2-keto acid decarboxylase (KDC) andan alcohol dehydrogenase (ADH) were used. kivd from Lactococcus lactiswas selected as the KDC for its high specificity towards 2-KIV and Adh2from Saccharomyces cerevisiae and yqhD from E. coli were both tested asthe ADH candidates for their different preference to cofactors NADH andNADPH, respectively. A plasmid containing kivd and either Adh2 or yqhDwas transformed into Ralstonia cells and tested for activity to convert2-KIV into isobutanol. Although the cells with kivd and Adh2 producedisobutanol from 2-KIV, the yqhD was a better alcohol dehydrogenase inRalstonia to produce isobutanol efficiently. Based on these result, yqhDwas shown to be more active for reducing isobutyaldehyde to isobutanol,because of the higher intracellular NADPH level than NADH in theRalstonia eutropha.

Using these two genes (kivd, yqhD), 5 different configurations wereconstructed for the expression of kivd and yqhD, either chromosomal orplasmid. After construction of strains, the efficiency of these enzymesexpressed in Ralstonia were measured by feeding experiment of 2-KIV.After 24 hr, the isobutanol production from 2-KIV was measured fromthese strains. As shown in FIG. 2D, the kivd-yqhD operons driven by CATgene promoter and phaP promoter were successful in converting 2-KIV intoisobutanol. The plasmid harboring Pcat promoter version of kivd-yqhDoperon was used for the construction of isobutanol production strain.

After construction of all the functionally expressed 5 genes needed forthe production of isobutanol from pyruvate, the various enzymes andoperons were engineered into one organism to construct an isobutanolproducing Ralstonia eutropha strain. LH67, which showed the strongestenzyme activities for alsS and ilvCD, was transformed with the plasmidharboring the most efficient kivd-yqhD operon with Pcat promoter. Thefinal strain, LH74, was tested for the production of isobutanol. In 5 Lfermentor operation, this strain was found to produce 120 mg/L ofisobutanol from fructose as carbon source in 40 hours. Interestingly,this strain also produced 180 mg/L of 3-Methyl-1-butanol, which is alsogood higher alcohol biofuel.

To test the electro-autotrophic production of isobutanol by R. eutrophastrain LH74, the strain was cultured in minimal media using 5 Lfermentor with autotrophic gas mixing condition (hydrogen, carbondioxide, and oxygen=10:1:1). Carbon dioxide is the only carbon sourceprovided in this fermentation. All gases were bubbled into the fermentorunder atmospheric pressure and the pH of the culture was held constantat 7.0. The produced higher alcohols were collected using chilledcondensing system from vent-gas line of fermentor. This fermentation wasrun over a 5.8 day period and produced a total 67.7 mg/L of isobutanolwith a final OD_(600nm) of 12.72 (OD_(436nm) higher than 20) (FIG. 2E).Both the OD and the isobutanol production continued to climb over theduration of the 5.8 day fermentation. The isobutanol production showedno signs of a plateau after 5.8 days. However, under these conditions,major carbon flow from CO₂ fixation via CBB pathway is still directedtoward cell mass production rather than biofuel production. Thisexperiment demonstrates isobutanol production in autotrophic conditionsusing R. eutropha indicating successful electro-autotrophic productionof higher alcohol.

From the intermediate 2-Ketoisovalerate (2-KIV) feeding experiment, thedata suggested that the activity of the keto acid decarboxylation andreductation part of the pathway (catalyzed by kivd and yqhD) may not bethe limiting factor of the production rate in vivo. Therefore, one ofthe hypotheses could be that the part of the pathway upstream of kivdand yqhD may be the bottleneck of isobutanol production in this strain.This part of the pathway overlaps with the native valine biosynthesispathway and was enhanced by overexpressing alsS (Bacillus subtilis),ilvC (Escherichia coli), and ilvD (Escherichia coli). Although theactivities of alsS, ilvC, and ilvD were measured in enzymatic assays andshown significant increased compared to wildtype strain, the absolutevalue of the enzymatic activity was lower than E. coli isobutanolproduction strains in other research. And because the alsS, ilvC, andilvD operon was integrated into the Ralstonia chromosome with only onecopy (LH74), it was reasoned that the relatively low activity of thispart of the pathway may be due to the low gene dosage in the strain.

To explore this possibility, alsS, ilvC, and ilvD were also put into amultiple copy plasmid in addition to kivd and yqhD. The whole operoncontaining all five genes of the pathway was driven by the pPhaPpromoter. After transforming this plasmid into wildtype Ralstonia cells,the resulted strain was able to produce around 200 mg/L isobutanol inone day in minimal medium with fructose as the substrate, which is overtwo fold of the amount produced by the previous strain in the samecondition. The final titer of isobutanol can reach around 500 mg/L inminimal medium with fructose, although in these experiments the cellgrowth was retarded and the production limited after two days,indicating toxicity of the production pathway caused by the high leveloverexpression from the multiple copy plasmid.

To overcome the toxicity effect while still maintaining the high genedosage conveyed by the plasmid system, the alsS from Bacillus subtilisis replaced by several acetohydroxy acid synthase (AHAS) genes fromdifferent organisms in the multiple copy plasmids and tested for theactivity and toxicity. The genes tested include ilvBN (E. coli), ilvIH(E. coli), and alsS (Klebsiella pneumoniae). The results showed thatdifferent AHAS proteins may have a broad range of activity in vivo,resulting in different isobutanol production rate and titer. Forexample, when alsS from Klebsiella pneumoniae is overexpressed, thecells were able to produce around 1.2 g/L isobutanol in minimal mediumwith fructose in one day as shown in FIG. 2F. However, although theAHASs tested vary in protein sequences and structures, all of themresulted in toxicity, indicating the toxicity of the pathway may not bedue to the protein expression or folding problem related to one specificAHAS protein.

For electro-produced formate as a single carbon source, conditions forautotrophic growth on formate were developed. Under standard minimalmedium (German medium) with formate, Ralstonia showed very poor growthas shown in FIG. 2G. To overcome this a buffered medium with HEPES wasused to control pH during growth. Using this growth condition, more thanOD_(436nm) 1 was grown in 2 days.

The examples set forth above are provided to give those of ordinaryskill in the art a complete disclosure and description of how to makeand use the embodiments of the devices, systems and methods of thedisclosure, and are not intended to limit the scope of what theinventors regard as their invention. Modifications of theabove-described modes for carrying out the invention that are obvious topersons of skill in the art are intended to be within the scope of thefollowing claims. All patents and publications mentioned in thespecification are indicative of the levels of skill of those skilled inthe art to which the invention pertains. All references cited in thisdisclosure are incorporated by reference to the same extent as if eachreference had been incorporated by reference in its entiretyindividually.

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

Submitted on Jan. 15, 2011 along with the e-filing of this applicationis a .txt file of a sequence listing which is incorporated herein byreference in its entirety.

1. A recombinant microorganism capable of using H₂ or formate forreduction of CO₂ and wherein the microorganism produces an alcoholselected from the group consisting of 1-propanol, isobutanol, 1-butanol,2-methyl 1-butanol, 3-methyl 1-butanol and 2-phenylethanol from CO₂ asthe carbon source, wherein the alcohol is produced from a metabolitecomprising a 2-keto acid.
 2. The recombinant microorganism of claim 1,wherein the microorganism has a naturally occurring H₂ and/or formatereduction pathway and at least one recombinant enzyme for the productionof an intermediate in the synthesis of the alcohol.
 3. The recombinantmicroorganism of claim 1, wherein the microorganism comprises expressionof a heterologous or overexpression of an endogenous carbon-fixationenzyme and heterologous or overexpression of a hydrogenase and/orformate dehydrogenase such that the microorganism can utilize H₂ and/orformate as a reducing metabolite.
 4. The recombinant microorganism ofclaim 1, 2, or 3, wherein the alcohol is isobutanol.
 5. The recombinantmicroorganism of claim 4, wherein the recombinant microorganism isobtained from a Ralstonia sp. parental organism.
 6. The recombinantmicroorganism of claim 2, wherein the recombinant microorganism isobtained from a Ralstonia sp. parental organism.
 7. The recombinantmicroorganism of claim 1, wherein the 2-keto acid is selected from thegroup consisting of 2-ketobutyrate, 2-ketoisovalerate, 2-ketovalerate,2-keto 3-methylvalerate, 2-keto 4-methyl-pentanoate, and phenylpyruvate.8. The recombinant microorganism of claim 1, 5 or 6 comprising elevatedexpression or activity of a 2-keto-acid decarboxylase and an alcoholdehydrogenase, as compared to a parental microorganism.
 9. Therecombinant microorganism of claim 8, wherein the 2-keto-aciddecarboxylase is selected from the group consisting of Pdc6, Aro10,Thi3, Kivd, and Pdc, or homolog thereof.
 10. The recombinantmicroorganism of claim 8, wherein the 2-keto-acid decarboxylase isencoded by a nucleic acid sequence derived from a gene selected from thegroup consisting of PDC6, ARO10, THI3, kivd, and pdc, or homologthereof.
 11. The recombinant microorganism of claim 10, wherein the2-keto-acid decarboxylase is encoded by a nucleic acid sequence derivedfrom the kivd gene, or homolog thereof.
 12. The recombinantmicroorganism of claim 8, wherein the alcohol dehydrogenase is Adh2, orhomolog thereof.
 13. The recombinant microorganism of claim 8, whereinthe 2-alcohol dehydrogenase is encoded by a nucleic acid sequencederived from the ADH2 gene, or homolog thereof.
 14. The recombinantmicroorganism of claim 1, wherein the microorganism is selected from agenus of Escherichia, Corynebacterium, Lactobacillus, Lactococcus,Salmonella, Enterobacter, Enterococcus, Erwinia, Pantoea, Morganella,Pectobacterium, Proteus, Serratia, Shigella, Klebsiella, Citrobacter,Saccharomyces, Dekkera, Klyveromyces, and Pichia.
 15. The recombinantmicroorganism of claim 14, wherein the biosynthetic pathway for theproduction of an amino acid in the organism is modified for productionof the alcohol.
 16. The recombinant microorganism of claim 14, whereinthe 2-keto acid is selected from the group consisting of 2-ketobutyrate,2-ketoisovalerate, 2-ketovalerate, 2-keto 3-methylvalerate, 2-keto4-methyl-pentanoate, and phenylpyruvate.
 17. The recombinantmicroorganism of claim 1, wherein the microorganism comprises reducedethanol production capability compared to a parental microorganism. 18.The recombinant microorganism of claim 1 or 14, wherein themicroorganism comprises a reduction or inhibition in the conversion ofacetyl-coA to ethanol.
 19. The recombinant microorganism of claim 1 or18, wherein the recombinant microorganism comprises a reduction of anethanol dehydrogenase thereby providing a reduced ethanol productioncapability.
 20. The recombinant microorganism of claim 18, wherein themicroorganism is derived from E. coli.
 21. The recombinant microorganismof claim 18, wherein the ethanol dehydrogenase is an adhE, homolog orvariant thereof.
 22. The recombinant microorganism of claim 21, whereinthe microorganism comprises a deletion or knockout of an adhE, homologor variant thereof.
 23. The recombinant microorganism of claim 1 or 14,wherein the microorganism comprises expression or elevated expression ofan enzyme that converts pyruvate to alpha-keto-isovalerate.
 24. Therecombinant microorganism of claim 23, wherein the enzyme is 2-keto-aciddecarboxylase.
 25. The recombinant microorganism of claim 1, comprisingelevated expression or activity of a 2-keto-acid decarboxylase and analcohol dehydrogenase, as compared to a parental microorganism.
 26. Therecombinant microorganism of claim 24 or 25, wherein the 2-keto-aciddecarboxylase is selected from the group consisting of Pdc, Pdc1, Pdc5,Pdc6, Aro10, Thi3, Kivd, and KdcA, a homolog or variant of any of theforegoing, and a polypeptide having at least 60% identity to any one ofthe foregoing and having 2-keto-acid decarboxylase activity.
 27. Therecombinant microorganism of claim 24 or 25, wherein the 2-keto-aciddecarboxylase is encoded by a polynucleotide having at least 60%identity to a nucleic acid selected from the group consisting of pdc,pdc1, pdc5, pdc6, aro10, thi3, kivd, kdcA, a homolog or variant of anyof the foregoing, or a fragment thereof and wherein the polynucleotideencodes a polypeptide having 2-keto acid decarboxylase activity.
 28. Therecombinant microorganism of claim 27, wherein the 2-keto-aciddecarboxylase is encoded by a polynucleotide derived from a kivd gene,or homolog thereof.
 29. The recombinant organism of claim 12, whereinthe alcohol dehydrogenase is selected from the group consisting of Adh1,Adh2, Adh3, Adh4, Adh5, Adh6, Sfa1, a homolog or variant of any of theforegoing, and a polypeptide having at least 60% identity to any one ofthe foregoing and having alcohol dehydrogenase activity.
 30. Therecombinant microorganism of claim 25, wherein the alcohol dehydrogenaseis encoded by a polynucleotide having at least 60% identity to a nucleicacid selected from the group consisting of an adh1, adh2, adh3, adh4,adh5, adh6, sfa1 gene, and a homolog of any of the foregoing and whereinthe polynucleotide encodes a protein having 2-alcohol dehydrogenaseactivity.
 31. The recombinant microorganism of claim 14, wherein therecombinant microorganism comprises one or more deletions or knockoutsin a gene encoding an enzyme that catalyzes the conversion of acetyl-coAto ethanol, catalyzes the conversion of pyruvate to lactate, catalyzesthe conversion of fumarate to succinate, catalyzes the conversion ofacetyl-coA and phosphate to coA and acetyl phosphate, catalyzes theconversion of acetyl-coA and formate to coA and pyruvate, condensationof the acetyl group of acetyl-CoA with 3-methyl-2-oxobutanoate(2-oxoisovalerate), isomerization between 2-isopropylmalate and3-isopropylmalate, catalyzes the conversion of alpha-keto acid tobranched chain amino acids, synthesis of Phe Tyr Asp or Leu, catalyzesthe conversion of pyruvate to acetyl-coA, catalyzes the formation ofbranched chain amino acids, catalyzes the formation ofalpha-ketobutyrate from threonine, catalyzes the first step inmethionine biosynthesis, and catalyzes the catabolism of threonine. 32.The recombinant microorganism of claim 1 or 31, wherein the recombinantmicroorganism comprises one or more gene deletions selected from thegroup consisting of adhE, ldhA, frdBC, fnr, pta, pflB, leuA, leuB, leuC,leuD, ilvE, tyrB, poxB, ilvB, ilvI, ilvA, metA, tdh, homologs of any ofthe foregoing and naturally occurring variants of any of the foregoing.33. The recombinant microorganism of claim 1, comprising a genotypeselected from the group consisting of: (a) a deletion or knockoutselected from the group consisting of ΔadhE, ΔldhA, Δpta, ΔleuA, ΔleuB,ΔleuC, ΔleuD, ΔpoxB, ΔilvB, ΔilvI, ΔmetA, Δtdh and any combinationthereof and comprising an expression or increased expression of kivd,ThrABC and adh2, wherein the microorganism produces 1-propanol; (b) adeletion or knockout selected from the group consisting of ΔadhE, ΔldhA,ΔfrdB, ΔfrdC, Δfnr, Δpta, ΔpflB, ΔleuA, ΔilvE, ΔpoxB, ΔilvA, and anycombination thereof and comprising an expression or increased expressionof kivd, ThrABC and adh2 wherein the microorganism produces isobutanol;(c) a deletion or knockout selected from the group consisting of ΔadhE,ΔldhA, Δpta, ΔpoxB, ΔilvB, ΔilvI, ΔmetA, Δtdh, and any combinationthereof and comprising an expression or increased expression of kivd,ThrABC and adh2 wherein the microorganism produces 1-butanol; and (d) adeletion or knockout selected from the group consisting of ΔadhE, ΔldhA,ΔfrdB, ΔfrdC, Δfnr, Δpta, ΔpflB, ΔilvE, ΔtyrB, and any combinationthereof and comprising an expression or increased expression of kivd,ThrABC and adh2 wherein the microorganism produces 3-methyl 1-butanol.34. The recombinant microorganism of any one of claims 1-33, wherein themicroorganism produces greater than 100 mg/L of isobutanol in 40 hoursfrom sugar.
 35. The recombinant microorganism of any one of claims 1-34,wherein the microorganism produces greater than 150 mg/L of3-methyl-1-butanol in 40 hours from sugar.
 36. The recombinantmicroorganism of claim 34 or 35, wherein the microorganism produces 120mg/L of isobutanol or 180 mg/L of 3-methyl-1-butanol.
 37. A method ofproducing a biofuel, comprising culturing a microorganism of any one ofclaim 1-36 under conditions and in the presence os a suitable carbonsource and reducing agent and isolating the biofuel.
 38. The method ofclaim 37, wherein the biofuel is isobutanol.
 39. The method of claim 37,wherein the reducing agent is formate or H2.
 40. The method of claim 37,wherein the microorganism is obtained from a Ralstonia sp. parentalorganism.